Phenyl amino pyrimidine compounds and uses thereof

ABSTRACT

The present invention relates to phenyl amino pyrimidine compounds which are inhibitors of protein kinases including JAK kinases. In particular the compounds are selective for JAK2 kinases. The kinase inhibitors can be used in the treatment of kinase associated diseases such as immunological and inflammatory diseases including organ transplants; hyperproliferative diseases including cancer and myeloproliferative diseases; viral diseases; metabolic diseases; and vascular diseases.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of copending U.S. Ser. No. 12/530,610filed 19 Mar. 2010, now allowed, which is the national phase of PCTApplication PCT/AU2008/000339 having an international filing date of 12Mar. 2008, and claims priority from U.S. Ser. No. 60/894,264 filed 12Mar. 2007 and U.S. Ser. No. 61/016,252 filed 21 Dec. 2007.

TECHNICAL FIELD

The present invention relates to phenyl amino pyrimidine compounds whichare inhibitors of protein kinases, including JAK kinases. In particularthe compounds are selective for JAK2 kinases. The kinase inhibitors canbe used in the treatment of kinase associated diseases such asimmunological and inflammatory diseases including organ transplants;hyperproliferative diseases including cancer and myeloproliferativediseases; viral diseases; metabolic diseases; and vascular diseases.

BACKGROUND ART

JAKs are kinases which phosphorylate a group of proteins called SignalTransduction and Activators of Transcription or STATs. Whenphosphorylated, STATs dimerize, translocate to the nucleus and activateexpression of genes which lead to, amongst other things, cellularproliferation.

The central role played by the JAK family of protein tyrosine kinases inthe cytokine dependent regulation of both proliferation and end functionof several important cell types indicates that agents capable ofinhibiting the JAK kinases are useful in the prevention andchemotherapeutic treatment of disease states dependent on these enzymes.Potent and specific inhibitors of each of the currently known four JAKfamily members will provide a means of inhibiting the action of thecytokines that drive immunological and inflammatory diseases.

Myeloproliferative disorders (MPD) include, among others, polycythemiavera (PV), primary myelofibrosis, thrombocythemia, essentialthrombocythemia (ET), idiopathic myelofibrosis (IMF), chronicmyelogenous leukemia (CML), systemic mastocystosis (SM), chronicneutrophilic leukemia (CNL), myelodisplastic syndrome (MDS) and systemicmast cell disease (SMCD). JAK2 is a member of the JAK family of kinasesin which a specific mutation (JAK2V617F) has been found in 99% ofpolycythemia vera (PV) patients and 50% of essential thrombocytopenia(ET) and idiopathic myelofibrosis (MF). This mutation is thought toactivate JAK2, giving weight to the proposition that a JAK2 inhibitorwill be useful in treating these types of diseases.

Asthma is a complex disorder characterized by local and systemicallergic inflammation and reversible airway obstruction. Asthmasymptoms, especially shortness of breath, are a consequence to airwayobstruction, and death is almost invariably due to asphyxiation. AirwayHyper Responsiveness (AHR), and mucus hyper secretion by goblet cellsare two of the principle causes of airway obstruction in asthmapatients. Intriguingly recent work in animal experimental models ofasthma has underscored the importance of IL-13 as a key player in thepathology of asthma. Using a specific IL-13 blocker, it has beendemonstrated that IL-13 acts independently of IL-4 and may be capable ofinducing the entire allergic asthma phenotype, without the induction ofIgE (i.e., in a non-atopic fashion). This and other models have pointedto an important second tier mechanism for elicitating thepathophysiology of asthma, that is not dependent on the production ofIgE by resident B-cells or the presence of eonisophils. A directinduction of AHR by IL-13, represents an important process that islikely to be an excellent target for intervention by new therapies. Acontemplated effect of a JAK2 inhibitor to the lungs would result in thesuppression of the local release of IL-13 mediated IgE production, andtherefore reduction in histaminine release by mast cells andeosinophils. This and other consequences of the absence of IL-13indicate that many of the effects of asthma may be alleviated throughadministration of a JAK2 inhibitor to the lungs.

Chronic Obstructive Pulmonary Disease (COPD) is a term which refers to alarge group of lung diseases which can interfere with normal breathing.Current clinical guidelines define COPD as a disease state characterizedby airflow limitation which is not fully reversible. The airflowlimitation is usually both progressive and associated with an abnormalinflammatory response of the lungs to noxious particles and gases,particularly cigarette smoke and pollution. Several studies have pointedto an association between increased production of IL-13 and COPD,lending support to the proposition that the potential alleviation ofasthma symptoms by use of a JAK2 inhibitor, may also be achieved inCOPD. COPD patients have a variety of symptoms including cough,shortness of breath, and excessive production of sputum. COPD includesseveral clinical respiratory syndromes including chronic bronchitis andemphysema.

Chronic bronchitis is a long standing inflammation of the bronchi whichcauses increased production of mucus and other changes. The patient'ssymptoms are cough and expectoration of sputum. Chronic bronchitis canlead to more frequent and severe respiratory infections, narrowing andplugging of the bronchi, difficult breathing and disability.

Emphysema is a chronic lung disease which affects the alveoli and/or theends of the smallest bronchi. The lung loses its elasticity andtherefore these areas of the lungs become enlarged. These enlarged areastrap stale air and do not effectively exchange it with fresh air. Thisresults in difficult breathing and may result in insufficient oxygenbeing delivered to the blood. The predominant symptom in patients withemphysema is shortness of breath.

Additionally, there is evidence of STAT activation in malignant tumors,among them lung, breast, colon, ovarian, prostate and liver cancer, aswell as Hodgkin's lymphoma, multiple myeloma and hepatocellularcarcinoma. Chromosomal translocations involving JAK2 fusions to Tel, Bcrand PCM1 have been described in a number of hematopoietic malignanciesincluding chronic myelogenous leukemia (CML), acute myelogenous leukemia(AML), chronic eosinophilic leukemia (CEL), myelodisplastic syndrome(MDS), myeloproliferative disease (MPD) and acute lymphocytic leukemia(ALL). This suggests treatment of hyperproliferative disorders such ascancers including multiple myeloma; prostate, breast and lung cancer;Hodgkin's Lymphoma; CML; AML; CEL; MDS; ALL; B-cell Chronic LymphocyticLeukemia; metastatic melanoma; glioma; and hepatoma, by JAK inhibitorsis indicated.

Potent inhibitors of JAK2, in addition to the above, will also be usefulin vascular disease such as hypertension, hypertrophy, cardiac ischemia,heart failure (including systolic heart failure and diastolic heartfailure), migraine and related cerebrovascular disorders, stroke,Raynaud's phenomenon, POEMS syndrome, Prinzmetal's angina, vasculitides,such as Takayasu's arteritis and Wegener's granulomatosis, peripheralarterial disease, heart disease and pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a pulmonary vascular diseaseaffecting the pulmonary arterioles resulting in an elevation inpulmonary artery pressure and pulmonary vascular resistance but withnormal or only mildly elevated left-sided filling pressures. PAH iscaused by a constellation of diseases that affect the pulmonaryvasculature. PAH can be caused by or associated with collagen vasculardisorders such as systemic sclerosis (scleroderma), uncorrectedcongenital heart disease, liver disease, portal hypertension, HIVinfection, Hepatitis C, certain toxins, splenectomy, hereditaryhemorrhagic teleangiectasia, and primary genetic abnormalities. Inparticular, a mutation in the bone morphogenetic protein type 2 receptor(a TGF-b receptor) has been identified as a cause of familial primarypulmonary hypertension (PPH). It is estimated that 6% of cases of PPHare familial, and that the rest are “sporadic.” The incidence of PPH isestimated to be approximately 1 case per 1 million population. Secondarycauses of PAH have a much higher incidence. The pathologic signature ofPAH is the plexiform lesion of the lung which consists of obliterativeendothelial cell proliferation and vascular smooth muscle cellhypertrophy in small precapillary pulmonary arterioles. PAH is aprogressive disease associated with a high mortality. Patients with PAHmay develop right ventricular (RV) failure. The extent of RV failurepredicts outcome. The JAK/STAT pathway has recently been implicated inthe pathophysiology of PAH. JAKs are kinases which phosphorylate a groupof proteins called Signal Transduction and Activators of Transcriptionor STATs. When phosphorylated, STATs dimerize, translocate to thenucleus and activate expression of genes which lead to proliferation ofendothelial cells and smooth muscle cells, and cause hypertrophy ofcardiac myocytes. There are three different isoforms of JAK: JAK1, JAK2,and JAK3. Another protein with high homology to JAKs is designated TYK2.An emerging body of data has shown that the phosphorylation of STAT3, asubstrate for JAK2, is increased in animal models of PAH. In the ratmonocrotaline model, there was increased phosphorylation of thepromitogenic transcription factor STAT3. In this same study pulmonaryarterial endothelial cells (PAECs) treated with monocrotaline developedhyperactivation of STAT3. A promitogenic agent or protein is an agent orprotein that induces or contributes to the induction of cellularproliferation. Therefore, one effect of JAK2 inhibition would be todecrease proliferation of endothelial cells or other cells, such assmooth muscle cells. A contemplated effect of a JAK2 inhibitor would beto decrease the proliferation of endothelial cells or other cells whichobstruct the pulmonary arteriolar lumen. By decreasing the obstructiveproliferation of cells, a JAK2 inhibitor could be an effective treatmentof PAH.

Additionally the use of JAK kinase inhibitors for the treatment of viraldiseases and metabolic diseases is indicated.

Although the other members of the JAK family are expressed byessentially all tissues, JAK3 expression appears to be limited tohematopoetic cells. This is consistent with its essential role insignalling through the receptors for IL-2, IL4, IL-7, IL-9 and IL-15 bynon-covalent association of JAK3 with the gamma chain common to thesemultichain receptors. Males with X-linked severe combinedimmunodeficiency (XSCID) have defects in the common cytokine receptorgamma chain (gamma c) gene that encodes a shared, essential component ofthe receptors of interleukin-2 (IL-2), IL-4, IL-7, IL-9, and IL-15. AnXSCID syndrome in which patients with either mutated or severely reducedlevels of JAK3 protein has been identified, suggesting thatimmunosuppression should result from blocking signalling through theJAK3 pathway. Gene Knock out studies in mice have suggested that JAK3not only plays a critical role in B and T lymphocyte maturation, butthat JAK3 is constitutively required to maintain T cell function. Takentogether with the biochemical evidence for the involvement of JAK3 insignalling events downstream of the IL-2 and IL-4 receptor, these humanand mouse mutation studies suggest that modulation of immune activitythrough the inhibition of JAK3 could prove useful in the treatment ofT-cell and B-cell proliferative disorders such as transplant rejectionand autoimmune diseases. Conversely undesired inhibition of JAK3 couldhave a devastating effect on the immune status of an individual treatedwith drug.

Although the inhibition of various types of protein kinases, targeting arange of disease states, is clearly beneficial, it has been to datedemonstrated that the identification of a compound which is selectivefor a protein kinase of interest, and has good “drug like” propertiessuch as high oral bioavailability, is a challenging goal. In addition,it is well established that the predictability of inhibition, orselectivity, in the development of kinase inhibitors is quite low,regardless of the level sequence similarity between the enzymes beingtargeted.

The challenges in developing therapeutically appropriate JAK2 inhibitorsfor use in treatment kinase associated diseases such as immunologicaland inflammatory diseases including organ transplants;hyperproliferative diseases including cancer and myeloproliferativediseases; viral diseases; metabolic diseases; and vascular diseasesinclude designing a compound with appropriate specificity which also hasgood drug-likeliness.

There is therefore a continuing need to design and/or identify compoundswhich specifically inhibit the JAK family of kinases, and particularlycompounds which may preferentially inhibit one of the JAK kinasesrelative to the other JAK kinases, particularly JAK2. There is a needfor such compounds for the treatment of a range of diseases.

DISCLOSURE OF THE INVENTION

In a first aspect, there is provided a compound of formula I

wherein

Q and Z are independently selected from N and CR¹;

n is 1, 2 or 3;

R¹ is independently selected from hydrogen, halogen, R², OR², OH, R⁴,OR⁴, CN, CF₃, (CH₂)˜N(R²)₂, NO₂, R²R⁴, SO₂R⁴, NR²SO₂R³, COR⁴, NR²COR³,CO₂H, CO₂R², NR²COR⁴, R²CN, R²CN, R²OH, R²OR³ and OR⁵R⁴; or

two R¹ substituents together with the carbons which they are attached toform an unsaturated 5 or 6 membered heterocyclyl;

R² is substituted or unsubstituted C₁₋₄alkyl or substituted orunsubstituted C₁₋₄ alkylene where up to 2 carbon atoms can be optionallyreplaced with CO, NR^(Y), CONR^(Y), S, SO₂ or O;

R³ is R², C₂₋₄alkenyl or substituted or unsubstituted aryl;

R⁴ is NH₂, NHR², N(R¹)₂, substituted or unsubstituted morpholino,substituted or unsubstituted thiomorpholino, substituted orunsubstituted thiomorpholino-1-oxide, substituted or unsubstitutedthiomorpholino-1, 1-dioxide, substituted or unsubstituted piperazinyl,substituted or unsubstituted piperidinyl, substituted or unsubstitutedpyridinyl, substituted or unsubstituted pyrrolidinyl, substituted orunsubstituted pyrrolyl, substituted or unsubstituted oxazolyl,substituted or unsubstituted imidazolyl, substituted or unsubstitutedtetrahydrofuranyl and substituted or unsubstituted tetrahydropyranyl;

R⁵ is substituted or unsubstituted C₁₋₄alkylene;

R⁶-R¹⁰ are independently selected from H, R^(X)CN, halogen, substitutedor unsubstituted C₁₋₄alkyl, OR¹, CO₂R¹, N(R¹)₂, NO₂, CON(R¹)₂,SO₂N(R^(Y))₂, N(SO₂R¹)₂, substituted or unsubstituted piperazinyl,N(R^(Y))SO₂R² and CF₃;

R^(x) is absent or substituted or unsubstituted C₁₋₆alkylene wherein upto 2 carbon atoms can be optionally replaced with CO, NSO₂R¹, NR^(Y),CONR^(Y), S, SO₂ or O;

R^(Y) is H or substituted or unsubstituted C₁₋₄alkyl; and

R¹¹ is selected from H, halogen, substituted or unsubstituted C₁₋₄alkyl, OR², CO₂R², CN, CON(R¹)₂ and CF₃,

or an enantiomer thereof, a prodrug thereof or a pharmaceuticallyacceptable salt thereof.

In a second aspect, there is provided a process for the preparation ofthe compound of formula I defined above which comprises the step ofcoupling a compound of formula II

wherein

Y and R¹¹ and n are as defined above and X is a leaving group withcompounds of formulae III and IV

wherein

n, Z, R¹ and R⁶-R¹⁰ are as defined above; and

M is B or a metal such as Sn, Zn or Mg.

The compounds of formula I are kinase inhibitors, preferably JAKinhibitors, more preferably JAK2 inhibitors. These compounds are usefulin the treatment of kinase associated diseases such as immunological andinflammatory diseases including organ transplants; hyperproliferativediseases including cancer and myeloproliferative diseases; viraldiseases; metabolic diseases; and vascular diseases.

In a third aspect, there is provided a pharmaceutical agent ormetabolites thereof comprising the compound of formula I defined above.

There is also provided use of the compound of formula I as apharmaceutical agent or metabolites thereof.

There is further provided the compound of formula I defined above foruse as a pharmaceutical agent or metabolites thereof.

In a fourth aspect, there is provided a kinase inhibitor comprising thecompound formula I defined above.

There is also provided use of the compound of formula I defined above asa kinase inhibitor.

There is further provided the compound of formula I defined above foruse as a kinase inhibitor.

In a fifth aspect, there is provided a compound of formula 1 definedabove for use as a pharmaceutical agent or metabolites thereof,preferably a kinase inhibitor, more preferably a JAK kinase inhibitor,most preferably a JAK2 selective inhibitor.

The compound of formula I may also be administered in the form of apharmaceutical composition together with a pharmaceutically acceptablecarrier.

In a sixth aspect, there is provided a pharmaceutical compositioncomprising the compound of formula I defined above and apharmaceutically acceptable carrier.

In one embodiment, the pharmaceutical composition also comprises one ormore additional therapeutic agents.

The compound of formula I may be contained within or attached to animplant, such as a drug eluting stent. For example, when the compound isused for the treatment of pulmonary arterial hypertension (PAH), thecompound may be contained within or attached to a pulmonary arterystent, which may act locally, or be released from the stent into thepulmonary circulation where the compound exerts its therapeutic activityin the pulmonary vasculature.

In a seventh aspect, there is provided an implant which comprises thecompound of formula I defined above.

In an eighth aspect, there is provided a method for the treatment ofkinase associated diseases such as immunological and inflammatorydiseases including organ transplants; hyperproliferative diseasesincluding cancer and myeloproliferative diseases; viral diseases;metabolic diseases; and vascular diseases which comprises administeringan effective amount of the compound of formula I or a pharmaceuticalcomposition defined above to a subject in need thereof.

There is also provided use of the compound of formula I or apharmaceutical composition as defined above in the manufacture of amedicament for the treatment of kinase associated diseases such asimmunological and inflammatory diseases including organ transplants;hyperproliferative diseases including cancer and myeloproliferativediseases; viral diseases; metabolic diseases; and vascular diseases.

There is further provided use of the compound of formula I or apharmaceutical composition as defined above in the treatment of kinaseassociated diseases such as immunological and inflammatory diseasesincluding organ transplants; hyperproliferative diseases includingcancer and myeloproliferative diseases; viral diseases; metabolicdiseases; and vascular diseases.

There is still further provided the compound of the formula I or apharmaceutical composition defined above for use in the treatment ofkinase associated diseases such as immunological and inflammatorydiseases including organ transplants; hyperproliferative diseasesincluding cancer and myeloproliferative diseases; viral diseases;metabolic diseases; and vascular diseases.

In a ninth aspect, there is provided a method of inhibiting a kinase ina cell comprising contacting the cell with the compound of formula Idefined above.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the amino acid sequence alignment of selected JAK Kinases.The sequences shown are j2h=JAK2 (SEQ ID NO:1), j1h=JAK1 (SEQ ID NO:2),j3h=JAK3 (SEQ ID NO:3), and tyk2=TYK2 (SEQ ID NO:4). The sequences arenumbered with position 1 starting at amino acid 833 of the JAK2 sequence(taken from Genbank sequence NP_004963) and ends at the C-terminal aminoacid. The sequences shown correspond to the C-terminal kinase domain.

FIG. 2 shows a flow cytometry analysis of STAT5 phosphorylation inuntreated erythroleukaemic cells (HEL 92.1.7) versus cells that havebeen treated with 0.25, 0.5, 1, or 2 μM Compound 3, or DMSO/STAT5py.After treatment the cells were stained with mouse monoclonalanti-STAT5(Y694) PE antibody and analyzed using fluorescence activatedcell sorting (FACS). The histograms are shaded according to the foldchange in median fluorescence relative to the isotype control (Isocontlane in clear outline).

FIG. 3 shows the effect of compound 3 on IL-3 induced STAT5phosphorylation in BaF3 cells. BaF3 cells were incubated with vehicleonly, increasing concentrations of compound 3 or a positive controlcompound. The Western blots were treated with a STAT5 phospho-specificantibody and exposed to film for 5 minutes (top blot) and 1 minute(middle blot). The bottom blot shows total STAT protein, regardless ofphosphorylation state. These blots clearly show a decrease in STAT5phosphorylation in IL-3 stimulated BaF3 Cells with increasedconcentrations of compound 3.

FIG. 4 shows the effect of compound 3 on STAT5 phosphorylation in HELcells. HEL cells were incubated with vehicle only, increasingconcentrations of compound 3 or a positive control compound. The Westernblots were treated with a STAT5 phosphospecific antibody and exposed tofilm for 5 minutes (top blot) and 1 minute (middle blot). The bottomblot shows total STAT protein, regardless of phosphorylation state.These blots clearly show a decrease in STAT5 phosphorylation in HELCells with increased concentrations of compound 3.

FIG. 5 shows the effect of treatment with compound 3 on growthhormone-stimulated insulin-like growth factor-1 (IGF-1) concentrationsin mouse plasma.

FIG. 6 shows the efficacy of orally administered compound 3 in asubcutaneous tumour model of Ba/F3 TelJAK2 cells in nude mice.

FIG. 7 shows dot plots that demonstrate STAT5 phosphorylation (y axis)plotted against the expression of CD71 (x axis) in erythroid cells fromthe bone marrow of a patient with JAK2 V617F positive ET, as well as theeffect of compound 3 on pYSTAT5. In this case, the negative control (A)shows only a small amount of pYSTAT5 staining that increasessignificantly after stimulation with erythropoietin (B) (the positivecontrol). Addition of compound 3 caused a dose-dependent increase ininhibition of pYSTAT5 as illustrated in (C). This is presented as thepercentage inhibition of the measured pYSTAT5 activity of the positivecontrol in the left panel and as an absolute shift in fluorescenceintensity in the whole erythroid population in the right panel.

MODES OF CARRYING OUT THE INVENTION

The present invention relates to compounds of formula I which inhibitkinases, in particular JAK kinases such as JAK2 and are useful in thetreatment of kinase associated diseases such as immunological andinflammatory diseases including organ transplants; hyperproliferativediseases including cancer and myeloproliferative diseases; viraldiseases; metabolic diseases; and vascular diseases.

Compounds

The present invention relates to compounds of formula I

wherein

Q and Z are independently selected from N and CR¹;

n is 1, 2 or 3;

R¹ is independently selected from hydrogen, halogen, R², OR², OH, R⁴,OR⁴, CN, CF₃, (CH₂)˜N(R²)₂, NO₂, R²R⁴, SO₂R⁴, NR²SO₂R³, COR⁴, NR²COR³,CO₂H, CO₂R², NR²COR⁴, R²CN, R²CN, R²OH, R²OR³ and OR⁵R⁴; or

two R¹ substituents together with the carbons which they are attached toform an unsaturated 5 or 6 membered heterocyclyl;

R² is substituted or unsubstituted C₁₋₄alkyl or substituted orunsubstituted C₁₋₄ alkylene where up to 2 carbon atoms can be optionallyreplaced with CO, NR^(Y), CONR^(Y), S, SO₂ or O;

R³ is R², C₂₋₄alkenyl or substituted or unsubstituted aryl;

R⁴ is NH₂, NHR², N(R¹)₂, substituted or unsubstituted morpholino,substituted or unsubstituted thiomorpholino, substituted orunsubstituted thiomorpholino-1-oxide, substituted or unsubstitutedthiomorpholino-1, 1-dioxide, substituted or unsubstituted piperazinyl,substituted or unsubstituted piperidinyl, substituted or unsubstitutedpyridinyl, substituted or unsubstituted pyrrolidinyl, substituted orunsubstituted pyrrolyl, substituted or unsubstituted oxazolyl,substituted or unsubstituted imidazolyl, substituted or unsubstitutedtetrahydrofuranyl and substituted or unsubstituted tetrahydropyranyl;

R⁵ is substituted or unsubstituted C₁₋₄alkylene;

R⁶-R¹⁰ are independently selected from H, R^(X)CN, halogen, substitutedor unsubstituted C₁₋₄alkyl, OR¹, CO₂R¹, N(R¹)₂, NO₂, CON(R¹)₂,SO₂N(R^(Y))₂, N(SO₂R¹)₂, substituted or unsubstituted piperazinyl,N(R^(Y))SO₂R² and CF₃;

R^(x) is absent or substituted or unsubstituted C₁₋₆alkylene wherein upto 2 carbon atoms can be optionally replaced with CO, NSO₂R¹, NR^(Y),CONR^(Y), S, SO₂ or O;

R^(Y) is H or substituted or unsubstituted C₁₋₄alkyl; and

R¹¹ is selected from H, halogen, substituted or unsubstituted C₁₋₄alkyl, OR², CO₂R², CN, CON(R¹)₂ and CF₃, or an enantiomer thereof, aprodrug thereof or a pharmaceutically acceptable salt thereof.

In one embodiment, the compound of formula I has the formula Ia:

wherein,

Q and Z are independently selected from N and CR¹;

R¹ is independently selected from H, halogen, R², OR², OH, R⁴, CN, CF₃,NO₂, R²R⁴, SO₂R⁴, NR²SO₂R³, COR⁴, CO₂H, CO₂R², NR²COR³, NR²COR⁴, R²CN,R²OH, R²OR³ and OR⁵R³; or

two R¹ substitutents together with the carbon atoms to which they areattached form an unsaturated N-containing 5 or 6-membered heterocyclyl;

R² is C₁₋₄alkyl, or C₁₋₄alkylene;

R³ is R², C₂₋₄alkenyl or aryl;

R⁴ is NH₂, NHR², N(R²)₂, morpholino, thiomorpholino,thiomorpholino-1-oxide, thiomorpholino-1, 1-dioxide, 4-carbonylmethylpiperazinyl, 4-methyl piperazinyl, 3- or 4-hydroxy piperidinyl, 4hydroxymethyl piperidinyl, 4-pyrrolidinyl piperidinyl, 4 or 5-methyloxazolyl, 4-hydroxy pyridinyl, 3-hydroxy pyrrolyl, 3-hydroxypyrrolidinyl, pyridinyl pyrazolyl or imidazolyl;

R⁵ is C₂₋₄alkylene;

R⁶-R⁹ are independently selected from H, R^(X)CN, halogen, substitutedor unsubstituted C₁₋₄alkyl, substituted or unsubstituted aryl, OR¹,CO₂R¹, N(R¹)₂, NO₂, CON(R¹)₂ and CON(R¹)₂;

R^(X) is substituted or unsubstituted C₁₋₄alkylene wherein up to 2carbon atoms can be optionally replaced with CO, NSO₂R¹, NR^(Y),CONR^(Y), SO, SO₂, or O;

R^(Y) is H or substituted or unsubstituted C₁₋₄alkyl; and

R¹ is selected from H, halogen, substituted or unsubstituted C₁₋₄ alkyl,OR², CO₂R², CN, CON(R¹)₂ and CF₃, or an enantiomer thereof, a prodrugthereof or a pharmaceutically acceptable salt thereof.

Preferably Q is N and Z is CR¹.

Preferably R¹ is hydrogen, morpholinyl, CH₂morpholinyl, C₁₋₄alkoxy,thiomorpholinyl, 3-hydroxypyrrolidinyl, iodo, fluoro, OH, 4-hydroxypiperidinyl, 4 hydroxymethyl piperidinyl, N-methyl piperidinyl,3-hydroxy piperidinyl, carbonyl 4-pyrrolidinyl piperidinyl,oxy-4-piperidinyl, 4-carbonylmethyl piperazinyl, 4-methyl piperzinyl,4-NHSO₂CH₃-piperidinyl, 4-oxy piperidinyl, imidazolyl CON(R¹)₂, CF₃ orR²OR³.

Preferably R⁶ is H or methyl.

Preferably R⁷ is H, methyl, methoxy, halogen such as chloro or hydroxy.

Preferably R⁸ is H, R^(X)CN such as CONHCN, CH₂NHCOCN, CN,CONHC(CH₃)₂CN, NCNSO₂CH₃, SO₂NHCH₂CN or N(SO₂CH₃)CH₂CN, OH, CO₂CH₂CH₃,CON(R¹)₂, N(R¹)₂ or CO₂R¹.

Preferably R⁹ is H, R^(X)CN such as CONHCN, CH₂NHCOCN or CH₂NHCN,methoxy halogen, OCF₃ or CF₃.

Preferably R¹ is H, halogen, substituted or unsubstituted C₁₋₄alkyl,OR², CO₂R², CN or CF₃, more preferably H, methyl, methoxy, Cl, Br, F orCO₂R², most preferably H or methyl.

In a preferred embodiment, the compound of formula I or Ia has theformula Ib:

wherein

Z is independently selected from N and CH;

R¹ is independently selected from H, halogen, OH, CONHR², CON(R²)₂, CF₃,R²OR², CN, morpholino, thiomorpholinyl, thiomorpholino-1, 1-dioxide,substituted or unsubstituted piperidinyl, substituted or unsubstitutedpiperazinyl, imidazolyl, substituted or unsubstituted pyrrolidinyl andC₁₋₄alkylene wherein the carbon atoms are optionally replaced withNR^(Y) and/or O substituted with morpholino, thiomorpholinyl,thiomorpholino-1, 1-dioxide, substituted or unsubstituted piperidinyl,substituted or unsubstituted piperazinyl, imidazolyl or substituted orunsubstituted pyrrolidinyl;

R² is substituted or unsubstituted C₁₋₄alkyl;

R^(Y) is H or substituted or unsubstituted C₁₋₄alkyl;

R⁸ is R^(X)CN;

R^(X) is substituted or unsubstituted C₁₋₄alkylene wherein up to 2carbon atoms can be optionally replaced with CO, NSO₂R¹, NR^(Y),CONR^(Y), SO, SO₂ or O;

R¹¹ is H or C₁₋₄alkyl,

or an enantiomer thereof, a prodrug thereof or a pharmaceuticallyacceptable salt thereof.

Examples of compounds of formula I include, but are not limited to, thefollowing:

Compound Exact No. Structure mass ¹H NMR LC-MS Name  1

404.18 ¹H NMR (300 MHz, d₆- DMSO): δ 9.49 (1H, s), 8.54 (1H, d, 5.0 Hz),8.27 (2H, d, J = 8.7 Hz), 8.10 (2H, d, J = 8.7 Hz), 7.66 (2H, d, J = 9.1Hz), 7.38 (1H, d, J = 5.0 Hz), 6.93 (2H, d, J = 8.7 Hz), 4.35 (2H, q, J= 6.9 Hz), 3.73 (4H, m), 3.04 (4H, m), 1.34 (3H, t, J = 6.9 Hz). m/z404.3 M⁺ ethyl 4-(2-(4- morpholinophenyl amino)pyrimidin- 4-yl)benzoate 2

414.18 ¹H NMR (300 MHz, d₆- DMSO): δ 9.46 (1H, s), 9.34 (1H, s), 8.60(1H, s), 8.53 (1H, d, J = 5.1 Hz), 8.32 (1H, d, J = 7.8 Hz), 7.99 (1H,d, J = 7.8 Hz), 7.67 (3H, m), 7.68 (1H, d, J = 5.1 Hz), 6.92 (2H, d, J =9.0 Hz), 4.37 (2H, brs), 3.74 (4H, m), 3.04 (4H, m). m/z 414.3 M⁺N-(cyanomethyl)- 3-(2-(4- morpholinophenyl amino)pyrimidin-4-yl)benzamide  3

414.18 ¹H NMR (300 MHz, d₆- DMSO): δ 9.47 (1H, s), 9.32 5.5 Hz), 8.54(1H, d, J = 5.0 Hz), 8.27 (2H, d, J = 8.7 Hz), 8.02 (2H, d, J = 8.2 Hz),7.67 (2H, d, J = 9.1 Hz), 7.41 (1H, d, J = 5.5 Hz), 6.93 (2H, d, J = 9.1Hz), 4.36 (2H, d, J = 5.5 Hz), 3.75 (4H, m), 3.05 (4H, m). m/z 415.3[M + H]⁺ N-(cyanomethyl)- 4-(2-(4- morpholinophenyl amino)pyrimidin-4-yl)benzamide  4

419.20 ¹H NMR (300 MHz, d₆- DMSO): δ 8.42 (1H, d, J = 5.2 Hz), 8.21 (2H,d, J = 8.4 Hz), 7.95 (2H, d, J = 8.4 Hz), 7.60 (2H, d, J = 9.0 Hz), 7.27(1H, d, J = 5.2 Hz), 7.27 (1H, d, J = 5.2 Hz), 6.98 (2H, d, J = 9.0 Hz),3.84 (4H, m), 3.73 (2H, t, J = 5.8 Hz), 3.53 (2H, t, J = 5.8 Hz), 3.11(4H, m). m/z 419.4 M⁺ N-(2- hydroxyethyl)-4- (2-(4- morpholinophenylamino)pyrimidin- 4-yl)benzamide  5

357.16 ¹H NMR (300 MHz, CDCl₃): δ 8.49 (d, J = 5.1 Hz, 1H), 8.37- 8.36(m, 1H), 8.28-8.25 (m, 1H), 7.78-7.75 (m, 1H), 7.63- 7.61 (m, 1H),7.57-7.54 (m, 2H), 7.09 (d, J = 4.8 Hz, 1H), 7.00-6.97 (m, 2H), 3.89 (t,J = 4.5 Hz, 4H), 3.16 (t, J = 4.9 Hz, 4H). m/z 356.8 M⁺ 3-(2-(4-morpholinophenyl amino)pyrimidin- 4-yl)benzonitrile  6

357.16 ¹H NMR (300 MHz, CDCl₃): δ 8.50 (d, J = 5.1 Hz, 1H), 8.15 (d, J =8.7 Hz, 2H), 7.78 (d, J = 8.7 Hz, 2H), 7.58-7.55 (m, 2H), 7.13-7.11 (m,1H), 7.01-6.98 (m, 2H), 3.90 (t, J = 4.5 Hz, 4H), 3.16 (t, J = 4.2 Hz,4H). m/z 356.8 M⁺ 4-(2-(4- morpholinophenyl ammo)pyrimidin-4-yl)benzonitrile  7

375.15 ¹H NMR (300 MHz, CDCl₃): δ 8.49 (d, J = 5.7 Hz, 1H), 8.36 (dd, J= 5.7, 2.4 Hz, 1H), 8.29 (m, 1H), 7.54 (d, J = 9.3 Hz, 1H), 7.33 (t, J =9.0 Hz, 1H), 7.10 (br. s, 1H), 7.05 (d, J = 5.1 Hz, 1H), 6.97 (d, J =8.7 Hz, 2H), 3.88 (t, J = 5.1 Hz, 4H), 3.15 (t, J = 5.4 Hz, 4H). m/z375.0 M⁺ 2-fluoro-5-(2-(4- morpholinophenyl amino)pyrimidin-4-yl)benzonitrile  8

380.13 ¹H NMR (300 MHz, CDCl₃): δ 8.53 (d, J = 5.4 Hz, 1H), 8.44 (dd, J= 5.7 Hz, 2.1 Hz, 1H), 8.29 (m, 1H), 7.34 (t, J = 8.7 Hz, 1H), 7.19 (br.s, 1H), 7.12 (d, J = 5.1 Hz, 1H), 7.00 (s, 2H), 3.92 (s, 6H), 3.85 (s,3H). m/z 379.9 M⁺ 2-fluoro-5-(2- (3,4,5- trimethoxyphenyl-amino)pyrimidin-4- yl)benzonitrile  9

373.15 ¹H NMR (300 MHz, CDCl₃): δ 8.39 (m, 1H), 8.23 (d, J = 1.8 Hz,1H), 8.11 (dd, J = 8.7, 2.1 Hz, 1H), 7.57-7.55 (m, 2H), 7.05-7.02 (m,2H), 7.01-6.90 (m, 2H), 3.89 (t, J = 4.5 Hz, (m, 1H), 3.15-3.13 (m, 4H).m/z 373.0 M⁺ 2-hydroxy-5-(2- (4- morpholinophenyl amino)pyrimidin-4-yl)benzonitrile 10

428.20 ¹H NMR (300 MHz, CDCl₃): δ 8.45 (1H, d, J = 5.0 Hz), 7.72 (1H, d,J = 1.6 Hz), 7.76 (1H, dd, J = 1.6, 8.0 Hz), 7.52 (3H, m), 7.14 (1H, s),6.91 (2H, d, J = 9.0 Hz), 6.77 (1H, d, J = 5.0 Hz), 6.67 (1H, t, J = 5.7Hz), 4.39 (2H, d, J = 5.7 Hz), 3.86 (4H, m), 3.11 (4H, m), 2.48 (3H, s).m/z 428.3 M⁺ N-(cyanomethyl)- 3-methyl-4-(2-(4- morpholinophenylamino)pyrimidin- 4-yl)benzamide 11

428.20 ¹H NMR (300 MHz, CDCl₃: d₄-MeOH): δ 8.42 (1H, d, J = 5.2 Hz),7.99 (1H, brs), 7.96 (1H, dd, J = 1.2, 8.1 Hz), 7.62 (2H, d, J = 9.2Hz), 7.53 (1H, d, J = 8.0 Hz), 7.19 (1H, d, J = 5.2 Hz), 6.99 (2H, d, J= 9.2 Hz), 4.33 (2H, s), 3.89 (4H, m), 3.15 (4H, m), 2.54 (3H, s). *Partially obscured by CHCl₃ signal. m/z 428.3 M⁺ N-(cyanomethyl)-2-methyl-4-(2-(4- morpholinophenyl amino)pyrimidin- 4-yl)benzamide 12

428.20 ¹H NMR (300 MHz, d₆- DMSO): δ 9.40 (1H, s), 8.78 (1H, dd, J =5.5, 5.9 Hz), 8.48 (1H, d, J = 5.5 Hz), 8.03 (2H, m), 7.67 (2H, d, J =9.1 Hz), 7.50 (1H, t, J = 7.8 Hz), 7.43 (1H, m), 7.29 (1H, d, J = 5.0Hz), 6.93 (2H, d, J = 9.1 Hz), 4.39 (2H, d, J = 5.9 Hz), 3.73 (4H, m),3.71 (2H, s), 3.04 (4H, m). m/z 428.2 M⁺ 2-cyano-N-(3-(2- (4-morpholinophenyl amino)pyrimidin- 4- yl)benzyl)acetamide 13

400.20 ¹H NMR (300 MHz, d₆- DMSO): δ 9.41 (1H, s), 8.47 (1H, d, J = 5.0Hz), 8.13 (1H, brs), 8.02 (1H, ddd, J = 1.8, 4.1, 5.0 Hz), 7.68 (2H, d,J = 9.1 Hz), 7.49 (2H, brd, J = 4.5 Hz), 7.31 (1H, d, J = 5.0 Hz), 6.92(2H, d, J = 9.1 Hz), 3.85 (2H, d, J = 5.9 Hz), 3.73 (4H, m), 3.63 (2H,d, J = 7.3 Hz), 3.03 (4H, m). m/z 400.1 M⁺ 2-(3-(2-(4- morpholinophenylamino)pyrimidin- 4- yl)benzylamino) acetonitrile 14

414.18 ¹H NMR (300 MHz, d₆- DMSO): δ 10.46 (1H, s), 9.41 (1H, s), 8.53(1H, s), 8.49 (1H, d, J = 5.5 Hz), 7.83 (1H, d, J = 7.8 Hz), 7.72 (2H,d, J = 9.1 Hz), 7.58 (1H, brd, J = 8.2 Hz). 7.48 (1H, dd, J = 7.8, 7.8Hz), 7.24 (1H, d, J = 5.0 Hz), 6.96 (2H, d, J = 9.1 Hz), 3.95 (2H, 3.73(4H, m), 3.04 (4H, m). m/z 414.3 M⁺ 2-cyano-N-(3-(2- (4-morpholinophenyl amino)pyrimidin- 4- yl)phenyl)acetamide 15

386.19 ¹H NMR (300 MHz, d₆- acetonitrile): δ 8.42 (1H, d, J = 5.0 Hz),7.72 (1H, br), 7.64 (2H, d, J = 9.1 Hz), 7.51-7.54 (2H, m), 7.37 (1H,dd, J = 7.8, 8.2 Hz), 7.20 (1H, d, J = 5.0 Hz), 6.98 (2H, m), 6.90 (1H,m), 5.04 (1H, t, J = 6.9 Hz), 4.22 (2H, d, J = 6.9 Hz), 3.79 (4H, m),3.08 (4H, m). m/z 386.2 M⁺ 2-(3-(2-(4- morpholinophenyl amino)pyrimidin-4- yl)phenylamino) acetonitrile 16

387.17 ¹H NMR (300 MHz, CDCl₃: δ 8.50 (d, J = 5.1 Hz, 1H), 7.79 (d, J =1.2 Hz, 1H), 7.67 (d, J = 8.0 Hz, 1H), 7.61 (dd, J = 1.4, 8.0 Hz, 1H),7.56 (d, J = 9.0 Hz, 2H), 7.12 (br. s, 1H), 7.10 (d, J = 5.4 Hz, 1H),6.95 (d, J = 9.0 Hz, 2H), 4.05 (s, 3H), 3.89 (m, 4H), 3.14 (m, 4H). m/z388.2 [M + H]⁺ 2-methoxy-4-(2- (4- morpholinophenyl amino)pyrimidin-4-yl)benzonitrile 17

466.21 ¹H NMR (300 MHz, d₆- DMSO): δ 9.47 (1H, s), 9.24 (1H, t, J = 5.9Hz), 8.52 (1H, d, J = 5.5 Hz), 8.51 (2H, m), 8.24 (2H, d, J = 8.2 Hz),8.05 (2H, d, J = 8.7 Hz), 7.66 (2H, d, J = 9.1 Hz), 7.39 (1H, d, J = 5.5Hz), 7.32 (2H, d, J = 5.9 Hz), 6.92 (2H, d, J = 9.1 Hz), 4.52 (2H, d, J= 5.9 Hz), 3.74 (4H, m), 3.04 (4H, m). m/z 467.1 [M + H]⁺ 4-(2-(4-morpholinophenyl amino)pyrimidin- 4-yl)-N-(pyridin- 4-ylmethyl)benzamide 18

466.21 ¹H NMR (300 MHz, d₆- DMSO): δ 9.45 (1H, s), 9.20 (1H, t, J = 5.9Hz), 8.57 (1H, d, J = 1.8 Hz), 8.52 (1H, d, J = 5.0 Hz), 8.46 (1H, dd, J= 1.8, 5.0 Hz), 8.23 (2H, d, J = 8.7 Hz), 8.03 (2H, d, J = 8.7 Hz), 7.74(1H, ddd, J = 1.8, 2.8, 7.8 Hz), 7.66 (2H, d, J = 9.1 Hz), 7.38 (1H, d,J = 5.0 Hz), 7.36 (1H, m), 6.92 (2H, d, J = 9.1 Hz), 4.52 (2H, d, J =5.9 Hz), 3.73 (4H, m), 3.04 (4H, m). m/z 467.1 [M + H]⁺ 4-(2-(4-morpholinophenyl amino)pyrimidin- 4-yl)-N-(pyridin- 3-ylmethyl)benzamide 19

391.12 ¹H NMR (300 MHz, CDCl₃): δ 8.51 (d, J = 5.1 Hz, 1H), 8.21 (s,1H), 8.02 (d, J = 9.6 Hz, 1H), 7.78 (d, J = 8.4 Hz, 1H), 7.53 (d, J =9.0 Hz, 2H), 7.16 (br. s, 1H), 7.09 (d, J = 5.1 Hz, 1H), 6.96 (d, J =9.0 Hz, 2H), 3.88 (m, 4H), 3.15 (m, 4H). m/z 391.3/393.3 M⁺2-chloro-4-(2-(4- morpholinophenyl amino)pyrimidin- 4-yl)benzonitrile 20

419.16 ¹H NMR (300 MHz, d₆- DMSO): δ 9.59 (1H, s), 9.32 (1H, t, J = 5.5Hz), 8.59 (1H, d, J = 5.0 Hz), 8.31 (2H, d, J = 8.7 Hz), 8.02 (2H, d, J= 8.7 Hz), 7.47 (1H, d, J = 5.0 Hz), 7.30 (2H, s), 4.34 (2H, d, J = 5.5Hz), 3.80 (6H, s) 2 × OMe, 3.63 (2H, s) OMe. m/z 420.3 [M + H]⁺N-(cyanomethyl)- 4-(2-(3,4,5- trimethoxyphenyl- amino)pyrimidin-4-yl)benzamide 21

459.23 ¹H NMR (300 MHz, d₆- DMSO): δ 9.45 (1H, s), 8.63 (1H, t, J = 5.9Hz), 8.51 (1H, d, J = 5.0 Hz), 8.21 (2H, d, J = 8.7 Hz), 7.99 (2H, d, J= 8.2 Hz), 7.65 (2H, d, J = 9.1 Hz), 7.37 (1H, d, J = 5.0 Hz), 6.92 (2H,d, J = 9.1 Hz), 3.99 (1H, m), 3.79 (1H, m), 3.73 (4H, m), 3.62 (1H, m),3.30 (2H, m),* 3.04 (4H, m), 1.97-1.76 (3H, m), 1.65-1.54 (1H, m). m/z460.4 [M + H]⁺ 4-(2-(4- morpholinophenyl amino)pyrimidin- 4-yl)-N-((tetrahydrofuran- 2-yl)methyl) benzamide * Partially overlapping withwater signal from solvent. 22

441.19 ¹H NMR (300 MHz, d₆- DMSO): δ 12.44 (1H, brs), 10.90 (1H, brs),9.46 (1H, s), 8.52 (1H, d, J = 5.0 Hz), 8.24 (2H, d, J = 8.2 Hz), 8.14(2H, d, J = 8.7 Hz), 7.47 (3H, brd, J = 9.1 Hz),* 7.40 (1H, d, J = 5.0Hz), 6.93 (2H, d, J = 9.1 Hz), 6.66 (1H, brs), 3.74 (4H, m), *Overlapping resonances 2H d and 1H m. m/z 442.3 [M + H]⁺ 4-(2-(4-morpholinophenyl amino)pyrimidin- 4-yl)-N-(1H- pyrazol-3- yl)benzamide23

442.21 ¹H NMR (300 MHz, d₆- DMSO): δ 9.47 (1H, s), 8.83 (1H, s), 8.53(1H, d, J = 5.0 Hz), 8.25 (2H, d, J = 8.7 Hz), 8.01 (2H, d, J = 8.2 Hz),7.66 (2H, d, J = 9.1 Hz), 7.39 (1H, d, J = 5.0 Hz), 6.92 (2H, d, J = 9.1Hz), 3.73 (4H, m), 3.04 (4H, m), 1.71 (6H, s). m/z 443.4 [M + H]⁺ N-(2-cyanopropan-2- yl)-4-(2-(4- morpholinophenyl amino)pyrimidin-4-yl)benzamide 24

414.18 ¹HNMR (500 MHz, d₆-DMSO): δ 9.58 (s, 1H), 9.33 (t, J = 5.5 Hz,1H), 8.59 (d, J = 5.0 Hz, 1H), 8.29 (d, J = 8.3 Hz, 2H), 8.02 (d, J =5.1 Hz, 2H), 7.63 (s, 1H), 7.47 (d, J = 5.5 Hz, 1H), 7.25 (m, 1H), 7.16(t, J = 8.0 Hz, 1H), 6.59 (dd, J = 8.0, 2.0 Hz, 1H), 4.36 (d, J = 5.5Hz, 2H), 3.77 (m, 4H), 3.13 (m, 4H). m/z 415.4 [M + H]⁺ N-(cyanomethyl)-4-(2-(3- morpholinophenyl amino)pyrimidin- 4-yl)benzamide 25

430.16 ¹H NMR (300 MHz, d₆- DMSO): δ 9.48 (s, 1H), 9.32 (t, J = 5.4 Hz,1H), 8.53 (d, J = 4.8 Hz, 1H), 8.26 (d, J = 8.7 Hz, 2H), 8.02 (d, J =8.7 Hz, 2H), 7.66 (d, J = 9.3 Hz, 2H), 7.40 (d, J = 5.1 Hz, 1H), 6.92(d, J = 9.0 Hz, 2H), 4.35 (d, J = 5.7 Hz, 2H), 3.41 (m, 4H), 2.70 (m,4H). m/z 431.3 [M + H]⁺ N-(cyanomethyl)- 4-(2-(4- thiomorpholino-phenylamino) pyrimidin-4- yl)benzamide 26

378.17 ¹H NMR (300 MHz, CDCl₃): δ 8.37 (1H, d, J = 5.4 Hz), 7.74 (1H, d,J = 1.5 Hz), 7.54-7.60 (3H, m), 6.98-7.07 (3H, m), 6.93 (2H, d, J = 8.7Hz), 5.89 (1H, bs), 4.00 (3H, s), 3.88 (4H, m), 3.13 (4H, m). m/z 378.4M⁺ 2-methoxy-4-(2- (4- morpholinophenyl amino)pyrimidin- 4-yl)phenol 27

392.16 LC- ESI- MS (method B): rt 6.4 min, m/z 393.1 [M + H]⁺1-(4-(4-(4-amino- 3- nitrophenyl) pyrimidin-2- ylamino)phenyl)pyrrolidin-3-ol 28

375.17 ¹H NMR (300 MHz, d₆- DMSO): δ 9.46 (s, 1H), 8.51 (d, J = 5.2 Hz,1H), 8.20 (d, J = 8.4 Hz, 2H), 8.07 (brs, 1H), 8.01 (d, J = 8.4 Hz, 2H),7.66 (d, J = 9.0 Hz, 2H), 7.47 (brs, 1H), 7.39 (d, J = 4.8 Hz, 1H), 6.92(d, J = 9.1 Hz, 2H), 3.73 (m, 4H), 3.04 (m, 4H). m/z 376.1 [M + H]⁺ andm/z 374.2 [M − H]⁻ 4-(2-(4- morpholinophenyl amino)pyrimidin-4-yl)benzamide 29

445.03 ¹H NMR (500 MHz, d₆- DMSO): δ 9.91 (s, 1H), 8.63 (d, J = 5.0 Hz,1H), 8.29 (d, J = 8.0 Hz, 2H), 8.11 (d, J = 8.5 Hz, 2H), 7.70 (d, J =9.0 Hz, 2H), 7.64 (d, J = 9.0 Hz, 2H), 7.51 (d, J = 4.5 Hz, 1H), 4.36(q, J = 7.0 Hz, 2H), 1.35 (t, J = 7.5 Hz, 3H). m/z found 446.2 [M + H]⁺ethyl 4-(2-(4- iodophenylamino) pyrimidin-4- yl)benzoate 30

455.02 ¹H NMR (300 MHz, d₆- DMSO): δ 9.88 (s, 1H), 9.61 (t, J = 5.4 Hz,1H), 8.61 (d, J = 5.1 Hz, 1H), 8.27 (d, J = 8.4 Hz, 2H), 8.09 (d, J =8.4 Hz, 2H), 7.7 (d, J = 9.0 Hz, 2H), 7.63 (d, J = 9.0 Hz, 2H), 7.52 (d,J = 5.1 Hz, 1H), 4.33 (d, J = 5.4 Hz, 2H). m/z 456.2 [M + H]⁺N-(cyanomethyl)- 4-(2-(4- iodophenylamino) pyrimidin-4- yl)benzamide 31

464.16 ¹H NMR (300 MHz, CDCl₃): δ 8.46 (d, J = 5.1 Hz, 1H), 8.15 (d, J =8.7 Hz, 2H), 7.65 (d, J = 8.7 Hz, 2H), 7.56 (d, J = 9.3 Hz, 2H), 7.16(bs, 1H), 7.09 (d, J = 5.4 Hz, 1H), 6.96 (d, J = 9.3 Hz, 2H), 4.00 (s,2H), 3.90- 3.87 (m, 4 H), 3.17-3.13 (m, 4H), 3.09 (s, 3H). m/z 465.4[M + H]⁺ N-(cyanomethyl)- N-(4-(2-(4- morpholinophenyl amino)pyrimidin-4- yl)phenyl)methane sulfonamide 32

425.15 ¹H NMR (300 MHz, d₆- DMSO): δ 9.36 (s,lH), 8.39 (s, 1H), 7.95 (d,J = 8.1 Hz, 2H), 7.84 (d, J = 8.1 Hz, 2H), 7.61 (m, 2H), 7.48 (s, 2H),6.88 (m, 2H), 3.72 (m, 4H), 3.01 (m, 4H), 2.19 (s, 3H). m/z 426.3 [M +H]⁺ 4-(5-methyl-2-(4- morpholinophenyl amino)pyrimidin- 4-yl)benzenesulfonamide 33

428.20 ¹H NMR (300 MHz, d₆- DMSO): δ 9.70 (s, 1H), 9.37- 9.31 (m, 1H),8.59 (d, J = 5.1 Hz, 1H), 8.29 (d, J = 8.7 Hz, 2H), 8.03 (d, J = 9.0 Hz,2H), 7.78 (d, J = 9.0 Hz, 2H), 7.47 (d, J = 5.1 Hz, 1H), 7.24 (d, J =9.0 Hz, 2H), 4.35 (d, J = 5.7 Hz, 2H), 3.64-3.50 (m, 4H), 3.41 (s, 2H),2.35 (brs, 4H). m/z 429.3 [M + H]⁺ N-(cyanomethyl)- 4-(2-(4-(morpholinomethyl) phenylamino) pyrimidin-4- yl)benzamide 34

471.20 ¹H NMR (300 MHz, d₆- DMSO): δ 9.49 (s, 1H), 9.00 (t, J = 6.0,1H), 8.67 (t, J = 5.4 Hz, 1H), 8.53 (d, J = 5.1 Hz, 1H), 8.26 (d, J =8.7 Hz, 2H), 8.05 (d, J = 9.0 Hz, 2H), 7.67 (d, J = 9.0 Hz, 2H), 7.41(d, J = 5.4 Hz, 1H), 6.94 (d, J = 9.0 Hz, 2H), 4.16 (d, J = 5.4 Hz, 2H),3.95 (d, J = 5.7 Hz, 2H), 3.78- 3.72 (m, 4H), 3.08- 3.02 (m, 4H). m/z472.4 [M + H]⁺ N-(2- (cyanomethylamino)- 2-oxoethyl)-4- (2-(4-morpholinophenyl amino)pyrimidin- 4-yl)benzamide 35

464.08 ¹H NMR (300 MHz, CDCl₃/CD₃OD) δ 8.46 (d, J = 5.1 Hz, 1H), 7.92(d, J = 1.5 Hz, 1H), 7.62 (dd, J = 1.8, J = 8.1 Hz, 1H), 7.41 (d, J =8.1 Hz, 1H), 7.40 (m, 1H), 7.15 (m, 2H), 7.15 (d, J = 5.4 Hz, 1H), 6.55(m, 1H), 4.05 (s, 3H), 3.47 (s, 6H). m/z 465.2 [M + H]⁺ N-(4-(2-(3-hydroxyphenylamino) pyrimidin-4- yl)-2- methoxyphenyl)- N-(methylsulfonyl) methanesulfonamide 36

428.20 ¹H NMR (300 MHz, d₆- DMSO): δ 9.44 (s, 1H), 9.38- 9.30 (m, 1H),8.52 (d, J = 5.1, 1H), 8.26 (d, J = 8.4 Hz, 2H), 8.02 (d, J = 8.7 Hz,2H), 7.61 (d, J = 9.3 Hz, 2H), 7.38 (d, J = 5.1 Hz, 1H), 6.91 (d, J =9.3 Hz, 2H), 4.69 (d, J = 4.2 Hz, 1H), 4.35 (d, J = 5.4 Hz, 2H),3.68-3.50 (m, 2H), 2.82-2.68 (m, 2H), 1.93-1.74 (m, 2H), 1.58-1.39 (m,2H). m/z 429.3 [M + H]⁺ N-(cyanomethyl)- 4-(2-(4-(4- hydroxypiperidin-1- yl)phenylamino) pyrimidin-4- yl)benzamide 37

405.18 ¹H NMR (300 MHz, d6- DMSO): δ 9.54 (s, 1H), 8.54 (d, J = 5.7 Hz,1H), 8.51 (d, J = 2.7 Hz, 1H), 8.25 (d, J = 8.1 Hz, 2H), 8.10 (d, J =7.8 Hz, 2H), 7.98 (dd, J = 9.0 Hz, 2.7, 1H), 7.40 (d, J = 5.4 Hz, 1H),6.87 (d, J = 9.0 Hz, 1H), 4.35 (q, J = 6.9 Hz, 2H), 3.74-3.68 (m, 4H),3.40-3.33 (m, 4H), 1.34 (t, J = 7.5 Hz, 3H). m/z 406.3 [M + H]⁺ ethyl4-(2-(6- morpholinopyridin- 3- ylamino)pyrimidin- 4-yl)benzoate 38

385.15 ¹H NMR (300 MHz, d₆- DMSO): δ 9.98 (s, 1H), 8.65 (d, J = 4.8 Hz,1H), 8.32 (d, J = 8.1 Hz, 2H), 8.17 (s, 1H), 8.12 (d, J = 8.7 Hz, 2H),7.97 (d, J = 9.0 Hz, 2H), 7.68 (s, 1H), 7.60 (d, J = 8.7 Hz, 2H), 7.52(d, J = 4.8 Hz, 1H), 7.09 (s, 1H), 4.36 (q, J = 6.6 Hz, 2H), 1.35 (t, J= 7.2, 3H). m/z 386.3 [M + H]⁺ ethyl 4-(2-(4-(1H- imidazol-1-yl)phenylamino) pyrimidin-4- yl)benzoate 39

456.23 ¹H NMR (300 MHz, CDCl₃): δ 8.30 (s, 3H), 7.89 (d, J = 8.2 Hz,2H), 7.73 (d, J = 8.2 Hz, 2H), 7.48 (d, J = 8.9 Hz, 2H), 6.94 (d, J =8.6 Hz, 2H), 6.49 (t, J = 6.1 Hz, 1H), 4.43 (d, J = 5.9 Hz, 2H), 3.63(d, J = 12.0 Hz, 2H), 3.55 (t, J = 5.7 Hz, 2H), 2.72-2.63 (m, 2H), 2.23(s, 3H), 1.85 (d, J = 13.2 Hz, 2H), 1.48-1.39 (m, 2H), 1.35- 1.31 (m,1H). m/z 457.4 [M + H]⁺ N-(cyanomethyl)- 4-(2-(4-(4- (hydroxymethyl)piperidin-1- yl)phenylamino)- 5- methylpyrimidin- 4-yl)benzamide 40

420.18 ¹H NMR (300 MHz, d₆- DMSO): δ 9.35 (s, 1H), 8.38 (s, 1H), 7.74(d, J = 8.1 Hz, 1H), 7.63 (d, J = 9.0 Hz, 2H), 7.36 (s, 1H), 7.26 (d, J= 7.8 Hz, 1H), 6.88 (d, J = 9.3 Hz, 2H), 3.87 (s, 3H), 3.72 (m, 4H),3.01 (m, 4H), 2.20 (s, 3H). m/z 421.4 [M + H]⁺ 2-methoxy-4-(5-methyl-2-(4- morpholinophenyl amino)pyrimidin- 4-yl)benzoic acid 42

442.21 ¹H NMR (300 MHz, CD₃OD): δ 8.44 (d, J = 5.4 Hz, 1H), 8.25 (d, J =8.4 Hz, 2H), 7.98 (d, J = 9.0 Hz, 2H), 7.60 (d, J = 9.0 Hz, 2H), 7.29(d, J = 5.4 Hz, 1H), 7.03 (d, J = 9.3 Hz, 2H), 4.53 (brs, 1H), 4.36 (s,2H), 3.68-3.60 (m, 2H), 3.46 (d, J = 6.3 Hz, 2H), 2.73-2.64 (m, 2H),1.92-1.82 (m, 2H), 1.68- 1.52 (m, 1H), 1.48-1.32 (m, 2H). m/z 443.4 [M +H]⁺ N-(cyanomethyl)- 4-(2-(4-(4- (hydroxymethyl) piperidin-1-yl)phenylamino) pyrimidin-4- yl)benzamide 43

444.19 ¹H NMR (300 MHz, d₆- DMSO): δ 9.78 (s, 1H), 9.37 (s, 1H), 8.46(d, J = 5.1 Hz, 1H), 8.16 (d, J = 8.1 Hz, 1H), 7.85 (d, J = 1.8 Hz, 1H),7.76 (dd, J = 8.1, 1.8 Hz, 1H), 7.68 (d, J = 9.3 Hz, 2H), 7.35 (d, J =5.4 Hz, 1H), 6.93 (d, J = 9.0 Hz, 2H), 4.06 (s, 2H), 3.98 (s, 3H), 3.74(m, 4H), 3.04 (m, 4H). m/z 445.3 [M + H]⁺ 2-cyano-N-(2- methoxy-4-(2-(4-morpholinophenyl amino)pyrimidin- 4- yl)phenyl)acetamide 44

427.21 ¹H NMR (300 MHz, CD₃OD): δ 8.46 (d, J = 5.1 Hz, 1H), 8.25 (d, J =8.7 Hz, 2H), 7.98 (d, J = 8.7 Hz, 2H), 7.66 (d, J = 9.0 Hz, 2H), 7.31(d, J = 5.4 Hz, 1H), 7.03 (d, J = 9.0 Hz, 2H), 4.36 (s, 2H), 3.38-3.33(m, 4H), 3.25-3.20 (m, 4H), 2.80 (s, 3H). m/z 428.4 [M + H]⁺N-(cyanomethyl)- 4-(2-(4-(4- methylpiperazin- 1- yl)phenylamino)pyrimidin-4- yl)benzamide 45

444.19 ¹H NMR (300 MHz, d₆- DMSO): δ 9.47 (s, 1H), 8.87 (br t, J = 5.4Hz, 1H), 8.54 (d, J = 5.1 Hz, 1H), 7.96 (d, J = 8.1 Hz, 1H), 7.90 (s,1H), 7.82 (d, J = 8.1 Hz, 1H), 7.67 (d, J = 8.7 Hz, 2H), 7.43 (d, J =5.1 Hz, 1H), 6.93 (d, J = 9.3 Hz, 2H), 4.32 (d, J = 5.4 Hz, 2H), 4.04(s, 3H), 3.74 (m, 4H), 3.04 (m, 4H). m/z 445.3 [M + H]⁺ N-(cyanomethyl)-2-methoxy-4-(2- (4- morpholinophenyl amino)pyrimidin- 4-yl)benzamide 46

442.21 ¹H NMR (300 MHz, CDCl₃): δ 8.31 (s, 1H), 7.89 (d, J = 8.7 Hz,2H), 7.72 (d, J = 8.2 Hz, 2H), 7.49 (d, J = 9.3 Hz, 2H), 6.94 (m, 3H),6.60 (t, J = 5.7 Hz, 1H), 4.42 (d, J = 6.1 Hz, 2H), 3.93 (m, 2H),3.24-3.20 (m, 2H), 3.07-3.01 (m, 4H), 2.23 (s, 3H), 1.55-2.00 (m, 2H,partially obscured by grease impurity). m/z 443.3 [M + H]⁺N-(cyanomethyl)- 4-(2-(4-(3- hydroxypiperidin- 1- yl)phenylamino)- 5-methylpyrimidin- 4-yl)benzamide 47

458.21 ¹H NMR (300 MHz, d₆- DMSO): δ 9.33 (s, 1H), 8.87 (t, J = 5.7 Hz,1H), 8.38 (s, 1H), 7.93 (d, J = 7.8 Hz, 1H), 7.63 (d, J = 9.0 Hz, 2H),7.41 (d, J = 1.2 Hz, 1H), 7.33 (dd, J = 7.8, 1.5 Hz, 1H), 6.88 (d, J =9.0 Hz, 2H), 4.32 (d, J = 5.7 Hz, 2H), 3.97 (s, 3H), 3.73 (m, 4H), 3.01(m, 4H), 2.21 (s, 3H). m/z 459.3 [M + H]⁺ N-(cyanomethyl)-2-methoxy-4-(5- methyl-2-(4- morpholinophenyl amino)pyrimidin-4-yl)benzamide 48

415.10 ¹H NMR (300 MHz, d₆- DMSO): δ 12.40 (m, 4H), 9.31 (s, 1H), 8.38(d, J = 5.4 Hz, 1H), 8.08 (s, 2H), 7.62 (d, J = 8.7 Hz, 2H), 7.27 (d, J= 5.4 Hz, 1H), 6.90 (d, J = 9.3 Hz, 2H), 6.07 (brs, 2H), 3.74 (m, 4H),3.04 (m, 4H), 2.80-2.63 (m, 8H). m/z 416.2/ 418.2/ 420.2 [M + H]⁺4-(4-amino-3,5- dichlorophenyl)- N-(4- morpholinophenyl) pyrimidin-2-amine•citrate 49

444.19 ¹H NMR (300 MHz, CDCl₃): δ 8.25 (s, 1H), 8.22 (d, J = 8.7 Hz,2H), 7.87 (d, J = 8.6 Hz, 2H), 7.52 (d, J = 9.0 Hz, 2H), 6.93 (d, J =9.2 Hz, 2H), 6.88 (brs, 1H), 6.45 (t, J = 5.7 Hz, 1H), 4.43 (d, J = 5.8Hz, 2H), 3.87 (t, J = 4.8 Hz, 4H), 3.87 (s, 3H), 3.12 (t, J = 4.8 Hz,4H). m/z 445.3 [M + H]⁺ N-(cyanomethyl)- 4-(5-methoxy-2- (4-morpholinophenyl amino)pyrimidin- 4-yl)benzamide 50

509.25 ¹H NMR (300 MHz, CD₃OD): δ 8.56 (d, J = 5.4 Hz, 1H), 8.29 (d, J =8.7 Hz, 2H), 8.00 (d, J = 8.7 Hz, 2H), 7.91 (d, J = 9.0 Hz, 2H),7.45-7.40 (m, 3H), 4.37 (s, 2H), 3.20-2.72 (m, 2H), 2.78-2.72 (m, 4H),2.56-2.42 (m, 1H), 2.12-1.92 (m, 2H), 1.90-1.84 (m, 5H), 1.60-1.42 (m,2H), 1.32-1.28 (brs, 1H). m/z 510.4 [M + H]⁺ N-(cyanomethyl)-4-(2-(4-(4- (pyrrolidin-1- yl)piperidine-1- carbonyl)phenyl-amino)pyrimidin-4- yl)benzamide 51

518.24 ¹H NMR (300 MHz, d₆- DMSO): δ 10.33 (s, 1H), 10.13 (t, J = 5.4Hz, 1H), 9.35 (d, J = 5.1 Hz, 1H), 9.07 (d, J = 8.7 Hz, 2H), 8.83 (d, J= 8.4 Hz, 2H), 8.49 (d, J = 8.5 Hz, 2H), 8.22 (d, J = 5.1 Hz, 1H), 8.16-8.02 (m, 5H), 7.73 (d, J = 9.3 Hz, 2H), 5.18 (d, J = 3.6 Hz, 2H),5.15-5.06 (m, 1H), 4.30 m/z 519.3 [M + H]⁺ 4-(2-(4-(1-benzylpiperidin-4- yloxy)phenylamino) pyrimidin-4-yl)- N- (cyanomethyl)benzamide (s, 2H), 3.55-3.42 (m, 2H), 3.10-2.95 (m, 2H), 2.80-2.67 (m,2H), 2.50-2.38 (m, 2H). 52

415.18 ¹H NMR (300 MHz, d₆- DMSO): δ 9.50 (s, 1H), 9.38- 9.32 (m, 1H),8.54 (d, J = 5.1 Hz, 2H), 8.24 (d, J = 8.4 Hz, 2H), 8.02 (d, J = 8.7 Hz,2H), 7.97 (dd, J = 9.0, 2.7 Hz, 1H), 7.42 (d, J = 5.4 Hz, 1H), 6.86 (d,J = 9.0 Hz, 1H), 4.35 (d, J = 5.4 Hz, 2H), 3.76-3.68 (m, 4H), 3.40-3.35(m, 4H). m/z 416.3 [M + H]⁺ N-(cyanomethyl)- 4-(2-(6- morpholinopyridin-3- ylamino)pyrimidin- 4-yl)benzamide 53

448.14 ¹H NMR (300 MHz, CDCl₃/CD₃OD): δ 8.41 (s, 1H), 7.98 (m, 4H), 7.56(d, J = 8.6 Hz, 2H), 6.95 (d, J = 8.6 Hz, 2H), 4.35 (s, 2H), 3.80 (t, J= 4.8 Hz, 4H), 3.08 (t, J = 4.8 Hz, 4H). m/z 449.3 [M + H]⁺4-(5-chloro-2-(4- morpholinophenyl amino)pyrimidin- 4-yl)-N-(cyanomethyl) benzamide 54

458.21 ¹H NMR (300 MHz, d₆- DMSO): δ 9.74 (s, 1H), 9.25 (s, 1H), 8.34(s, 1H), 8.10 (d, J = 8.1 Hz, 1H), 7.64 (d, J = 9.0 Hz, 2H), 7.39 (s,1H), 7.28 (d, J = 8.1 Hz, 1H), 6.87 (d, J = 8.7 Hz, 2H), 4.05 (s, 2H),3.92 (s, 3H), 3.73 (m, 4H), 3.01 (m, 4H), 2.24 (s, 3H). m/z 459.4 [M +H]⁺ 2-cyano-N-(2- methoxy-4-(5- methyl-2-(4- morpholinophenylamino)pyrimidin- 4- yl)phenyl)acetamide 55

455.21 ¹H NMR (300 MHz, d₆- DMSO): δ 9.49 (s, 1H), 9.34- 9.28 (m, 1H),8.54 (d, J = 5.4 Hz, 1H), 8.26 (d, J = 8.7 Hz, 2H), 8.02 (d, J = 8.7 Hz,2H), 7.67 (d, J = 9.0 Hz, 2H), 7.41 (d, J = 5.1 Hz, 1H), 6.96 (d, J =9.0 Hz, 2H), 4.35 (d, J = 5.4 Hz, 2H), 3.62-3.54 (m, 4H), 3.11-3.00 (m,4H), 2.04 (s, 3H). m/z 456.3 [M + H]⁺ 4-(2-(4-(4- acetylpiperazin-1-yl)phenylamino) pyrimidin-4-yl)-N- (cyanomethyl) benzamide 56

519.21 LC- ESI- MS (method B): rt 5.8 min, m/z 520.3 [M + H]⁺N-(cyanomethyl)- 4-(5-methyl-2-(4- (4- (methylsulfonamido) piperidin-1-yl)phenylamino) pyrimidin-4- yl)benzamide 57

463.17 ¹H NMR (300 MHz, d₆- DMSO): δ 9.78 (brs, 1H), 8.55 (d, J = 5.4Hz, 1H), 8.22 (d, J = 8.7 Hz, 2H), 7.81 (brd, J = 8.7 Hz, 2H), 7.64 (d,J = 8.7 Hz, 2H), 7.48 (d, J = .8 Hz, 2H), 7.44 (d, J = 5.1 Hz, 1H), 7.29(brd, J = 8.1 Hz, 2H), 7.11 (ap. d, J = 7.8 Hz, 2H), 4.58 (d, J = 2.4Hz, 2H), 3.86 (m, 4H), 3.41 (t, J = 2.4 Hz, 1H), 3.34 (brm, 4H), 3.13(s, 3H), 2.28 (s, 3H). m/z 464.0 [M + H]⁺ N-(4-(2-(4- morpholinophenylamino)pyrimidin- 4-yl)phenyl)-N- (prop-2- ynyl)methane- sulfnamidetosylate 58

442.21 ¹H NMR (300 MHz, CD₃OD): δ 8.27 (s, 1H), 7.95 (d, J = 8.4 Hz,2H), 7.75 (d, J = 8.7 Hz, 2H), 7.52 (d, J = 9.1 Hz, 2H), 6.94 (d, J =9.0 Hz, 2H), 4.34 (s, 2H), 3.78-3.68 (m, 1H), 3.45-3.41 (m, 2H),2.80-2.75 (m, 2H), 2.21 (s, 3H), 1.93-1.91 (m, 2H), 1.65-1.62 (m, 2H).m/z 443.3 [M + H]⁺ N-(cyanomethyl)- 4-(2-(4-(4- hydroxypiperidin- 1-yl)phenylamino)- 5- methylpyrimidin- 4-yl)benzamide 59

428.20 ¹H NMR (300 MHz, d₆- DMSO): δ 9.51 (s, 1H), 9.36- 9.30 (m, 1H),8.54 (d, J = 5.1 Hz, 1H), 8.26 (d, J = 8.7 Hz, 2H), 8.03 (d, J = 8.4 Hz,2H), 7.68 (d, J = 9.0 Hz, 2H), 7.41 (d, J = 5.4 Hz, 1H), 6.93 (d, J =10.2 Hz, 2H), 4.35 (d, J = 5.4 Hz, 2H), 4.32-4.26 (m, 1H), 3.00-1.35 (m,9H). m/z 429.3 [M + H]⁺ N-(cyanomethyl)- 4-(2-(4-(piperidin- 4-yloxy)phenylamino) pyrimidin-4- yl)benzamide 60

482.17 ¹H NMR (300 MHz, d₆- DMSO): δ 10.11 (1H, brs), 9.64 (1H, s), 9.27(1H, brd, J = 5.0 Hz), 8.48 (1H d, J = 5.0 Hz), 8.33 (1H, d, J = 2.8Hz), 8.20 (2H, d, J = 8.9 Hz), 7.85 (1H, dd, J = 2.8, J = 8.7 Hz), 7.35(3H, m), 7.22 (1H, d, J = 8.9 Hz), 3.75 (4H, m), 3.07 (3H, s), 2.87 (7H,m). m/z 483.3 [M + H]⁺ N-methyl-5-(4-(4- (methylsulfonamido) phenyl)pyrimidin-2- ylamino)-2- morpholino- benzamide 61

486.20 ¹H NMR (300 MHz, CDCl₃): δ 8.97 (s, 1H), 7.85 (d, J = 8.3 Hz,2H), 7.63 (d, J = 8.3 Hz, 2H), 7.52 (d, J = 8.9 Hz, 2H), 7.36 (brs, 1H),6.92 (d, J = 9.0 Hz, 2H), 6.51 (t, J = 5.1 Hz, 1H), 4.41 (d, J = 5.8 Hz,2H), 4.18 (q, J = 7.2 Hz, 2H), 3.87 (t, J = 4.8 Hz, 4H), 3.14 (t, J =4.8 Hz, 4H), 1.16 (t, J = 7.1 Hz, 3H) m/z 487.3 [M + H]⁺ ethyl 4-(4-(cyanomethyl- carbamoyl)phenyl)-2- (4- morpholinophenyl amino)pyrimidine-5- carboxylate 62

499.23 ¹H NMR (300 MHz, d₆- DMSO): δ 9.33 (t, J = 5.4 Hz, 1H), 8.65 (d,J = 5.7 Hz, 1H), 8.44 (d, J = 7.8 Hz, 1H), 8.32- 8.29 (m, 3H), 8.12 (d,J = 7.8 Hz, 1H), 8.02 (d, J = 8.7 Hz, 2H), 7.60-7.53 (m, 3H), 4.36 (d, J= 5.1 Hz, 2H), 3.96 (s, 3H), 3.80-3.67 (m, 1H), 2.81- 2.77 (m, 2H), 2.18(s, 3H), 2.00-1.90 (m, 2H), 1.80 (m, 2H), 1.67-1.53 (m, 2H). m/z 500.4[M + H]⁺ 4-(4-(4- (cyanomethyl- carbamoyl)phenyl) pyrimidin-2-ylamino)-3- methoxy-N-(1- methylpiperidin-4- yl)benzamide 63

413.19 ¹H NMR (300 MHz, d₆- DMSO): δ 9.23 (t, J = 5.4 Hz, 1H), 8.86 (s,1H), 8.18 (d, J = 6.0 Hz, 1H), 8.00 (d, J = 8.1 Hz, 2H), 7.80 (d, J =8.7 Hz, 2H), 7.53 (d, J = 9.0 Hz, 2H), 7.03-6.99 (m, 2H), 6.90 (d, J =9.0 Hz, 2H), 4.34 (d, J = 5.4 Hz, 2H), 3.76-3.72 (m, 4H), 3.05-3.01 (m,4H). m/z 414.3 [M + H]⁺ N-(cyanomethyl)- 4-(2-(4- morpholinophenylamino)pyridin-4- yl)benzamide 65

492.09 ¹H NMR (300 MHz, CD₃OD/d₆- DMSO): δ 8.58 (s, 1H), 8.01 (s, 1H),7.99 (d, J = 8.3 Hz, 2H), 7.89 (d, J = 8.6 Hz, 2H), 7.58 (d, J = 8.7 Hz,2H), 6.92 (d, J = 9.2 Hz, 2H), 4.35 (s, 2H), 3.80 (t, J = 4.8 Hz, 4H),3.08 (t, J = 4.8 Hz, 4H) m/z 493.2 [M + H]⁺ 4-(5-bromo-2-(4-morpholinophenyl amino)pyrimidin- 4-yl)-N- (cyanomethyl) benzamide 66

485.22 ¹H NMR (300 MHz, d₆- DMSO): δ 9.71 (s, 1H), 9.38- 9.33 (m, 1H),8.58 (d, J = 5.4 Hz, 1H), 8.28 (d, J = 8.1 Hz, 2H), 8.02 (d, J = 8.7 Hz,2H), 7.77 (dd, J = 8.4 Hz, 1H), 7.68 (d, J = 2.4 Hz, 1H), 7.47 (d, J =4.8 Hz, 1H), 7.07 (d, J = 8.4 Hz, 1H), 4.35 (d, J = 5.4 Hz, 2H),3.70-3.63 (m, 4H), 3.12- 2.98 (m, 5H), 2.82 (s, 3H), 2.80-2.66 (m, 2H).m/z 486.4 [M + H]⁺ 5-(4-(4- (cyanomethyl- carbamoyl)phenyl) pyrimidin-2-ylamino)-N,N- dimethyl-2- morpholino- benzamide 67

513.25 ¹H NMR (300 MHz, d₆- DMSO): δ 9.79 (s, 1H), 9.53 (s, 1H),9.38-9.33 (m, 1H), 8.61 (d, J = 5.1 Hz, 1H), 8.45 (d, J = 2.7 Hz, 1H),8.35 (d, J = 8.7 Hz, 2H), 8.02 (d, J = 8.4 Hz, 2H), 7.85 (dd, J = 8.7, J= 2.7 Hz, 1H), 7.50 (d, J = 5.4 Hz, 1H), 7.30 (d, J = 8.4 Hz, 1H), 4.36(d, J = 5.4 Hz, 2H), 3.80- 3.73 (m, 4H), 2.94-2.88 (m, 4H), 1.44 (s,9H). m/z 514.3 [M + H]⁺ N-tert-butyl-5-(4- (4- (cyanomethyl-carbamoyl)phenyl) pyrimidin-2- ylamino)-2- morpholino- benzamide 68

485.22 ¹H NMR (300 MHz, d₆- DMSO): δ 9.78 (s, 1H), 9.60- 9.45 (m, 1H),9.37-9.32 (m, 1H), 8.59 (d, J = 5.1 Hz, 1H), 8.41 (d, J = 8.4, 2.7 Hz,1H), 8.35 (d, J = 8.4 Hz, 2H), 8.03 (d, J = 8.4 Hz, 2H), 7.83 (dd, J =2.7, 8.7 Hz, 1H), 7.50 (d, J = 5.1 Hz, 1H), 7.26 (d, J = 8.7 Hz, 1H),4.35 (d, J = 5.4 Hz, 2H), 3.80-3.73 (m, 4H), 3.44- 3.30 (m, 2H),2.92-2.88 (m, 4H), 1.20 (t, J = 7.2 Hz, 3H). m/z 486.3 [M + H]⁺ 5-(4-(4-(cyanomethyl- carbamoyl)phenyl) pyrimidin-2- ylamino)-N-ethyl- 2-morpholino- benzamide 69

432.17 ¹H NMR (300 MHz, d₆- DMSO): δ 9.77 (brs, 1H), 9.33 (t, J = 5.4Hz, 1H), 8.60 (d, J = 5.1 Hz, 1H), 8.27 (d, J = 8.4 Hz, 2H), 8.04 (d, J= 8.7 Hz, 2H), 7.78 (dd, J = 15.6, 2.4 Hz, 1H), 7.47-7.53 (m, 2H), 7.06-6.99 (m, 1H), 4.36 (d, J = 5.7 Hz, 2H), 3.76-3.72 (m, 4H), 2.98-2.94 (m,4H). m/z 433.3 [M + H]⁺ N-(cyanomethyl)- 4-(2-(3-fluoro-4-morpholinophenyl amino)pyrimidin- 4-yl)benzamide 70

448.14 ¹H NMR (300 MHz, CD₃OD/d₆- DMSO): δ 8.33 (d, J = 5.4 Hz, 1H),8.18 (m, 1H), 7.99-7.84 (m, 2H), 7.52 (d, J = 9.2 Hz, 2H), 7.16 (d, J =5.1 Hz, 1H), 6.88 (d, J = 9.1 Hz, 2H), 3.73 (t, J = 4.8 Hz, 4H), 3.01(t, J = 4.8 Hz, 4H). m/z 449.3 [M + H]⁺ N-(2-chloro-4-(2- (4-morpholinophenyl amino)pyrimidin- 4-yl)phenyl)-2- cyanoacetamide 71

498.16 ¹H NMR (300 MHz, d₆- DMSO): δ 9.43 (s, 1H), 8.50 (d, J = 5.1 Hz,1H), 8.20 (s, 1H), 8.16 (s, 2H), 7.64 (d, J = 9.1 Hz, 2H), 7.36 (d, J =5.1 Hz, 1H), 6.91 (d, J = 9.1 Hz, 2H), 4.04 (brs, 1H), 3.74 (t, J = 4.8Hz, 4H), 3.45 (m, 2H, obscured by water signal), 3.04 (t, J = 4.8 Hz,4H). m/z 499.2 [M + H]⁺ 2-cyano-N-(4-(2 (4- morpholinophenylamino)pyrimidin- 4-yl)-2- (trifluoromethoxy) phenyl)acetamide 72

482.17 ¹H NMR (300 MHz, d₆- DMSO): δ 10.02 (s, 1H), 9.40- 9.32 (m, 1H),8.64 (d, J = 5.1 Hz, 1H), 8.46 (brs, 1H), 8.30 (d, J = 8.7 Hz, 2H), 8.04(d, J = 8.1 Hz, 2H), 7.96 (dd, J = 9.3, 1.8 Hz, 1H), 7.59-7.54 (m, 2H),4.36 (d, J = 5.4 Hz, 2H), 2.76-2.66 (m, 4H), 2.85-2.80 (m, 4H). m/z483.3 [M + H]⁺ N-(cyanomethyl)- 4-(2-(4- morpholino-3- (trifluoromethyl)phenylamino) pyrimidin-4- yl)benzamide 73

578.24 ¹H NMR (300 MHz, d₆- DMSO): δ 9.89 (1H, t, J = 5.2 Hz), 9.77 (1H,s), 8.57 (1H, d, J = 5.5 Hz), 8.48 (1H, d, J = 2.7 Hz), 8.32 (2H, d, J =9.1 Hz), 7.91 (1H, dd, J = 2.7, 8.7 Hz), 7.62 (2H, d, J = 8.7 Hz), 7.45(1H, d, J = 5.5 Hz), 7.33 (1H, d, J = 8.7 Hz), 4.96 (2H, s), 3.80 (4H,m), 3.45 (2H, m), 3.20 (3H, s), 2..88 (4H, m), 2.45 (2H, m), 2.21 (6H,s). m/z 579.4 [M + H]⁺ 5-(4-(4-(N- (cyanomethyl) methylsulfonamido)phenyl)pyrimidin- 2-ylamino)-N-(2- (dimethylamino) ethyl)-2- morpholino-benzamide 74

532.15 ¹H NMR (300 MHz, d₆- DMSO): δ 9.97 (1H, s), 8.62 (1H, d, J = 5.0Hz), 8.36 (1H, d, J = 2.2 Hz), 8.26 (2H, d, J = 8.7 Hz), 8.02 (1H, dd, J= 2.3, J = 8.7 Hz), 7.63 (2H, d, J = 8.7 Hz), 7.54 (1H, d, J = 8.7 Hz),7.51 (1H, d, J = 5.1 Hz), 4.97 (2H, s), 3.69 (4H, m), 3.21 (3H, s), 2.82(4H, m). m/z 533.3 [M + H]⁺ N-(cyanomethyl)- N-(4-(2-(4- morpholino-3-(trifluoromethyl) phenylamino) pyrimidin-4- yl)phenyl)methanesulfonamide 75

401.19 ¹H NMR (300 MHz, d₆- DMSO): δ 9.73 (s, 1H), 9.34 (t, J = 4.8 Hz,1H), 8.60 (d, J = 5.1 Hz, 1H), 8.30 (d, J = 8.7 Hz, 2H), 8.03 (d, J =8.4 Hz, 2H), 7.91 (brs, 1H), 7.69 (brs, J = 4.3 Hz, 1H), 7.49 (d, J =5.4 Hz, 1H), 7.28 (t, J = 7.8 Hz, 1H), 6.93 (d, J = 7.8 Hz, 1H), 4.46(s, 2H), 4.35 (d, J = 5.1 Hz, m/z 402.3 [M + H]⁺ N-(cyanomethyl)-4-(2-(3- (propoxymethyl) phenylamino) pyrimidin-4- yl)benzamide 2H),3.42 (t, J = 6.6 Hz, 2H), 1.56 (m, 2H), 0.88 (t, J = 7.5 Hz, 3H). 76

482.17 ¹H NMR (300 MHz, d₆- DMSO): δ 10.16 (s, 1H), 9.50 (s, 1H), 8.54(d, J = 5.0 Hz, 1H), 8.52 (d, J = 2.5 Hz, 1H), 8.42 (dd, J = 8.0, 2.0Hz, 1H), 7.73 (d, J = 8.5 Hz, 1H), 7.63 (d, J = 9.0 Hz, 2H), 7.43 (d, J= 5.0 Hz, 1H), 6.92 (d, J = 9.0 Hz, 2H), 3.98 (s, 2H), 3.74 (t, J = 4.5Hz, 4H), 3.04 (t, J = 5.0 Hz, 4H). m/z 483.3 [M + H]⁺ 2-cyano-N-(4-(2-(4- morpholinophenyl amino)pyrimidin- 4-yl)-2- (trifluoromethyl)phenyl)acetamide 77

369.13 ¹H NMR (300 MHz, d₆- DMSO): δ 12.92 (s, 1H), 9.70 (s, 1H), 9.35(t, J = 5.6 Hz, 1H), 8.59 (d, J = 5.1 Hz, 1H), 8.30 (d, J = 8.4 Hz, 3H),8.04 (d, J = 8.4 Hz, 3H), 7.64 (m, 1H), 7.47 (m, 2H), 4.36 (d, J = 5.7Hz, 2H). m/z 370.3 [M + H]⁺ 4-(2-(1H-indazol- 5- ylamino)pyrimidin-4-yl)-N- (cyanomethyl) benzamide 78

440.20 ¹H NMR (300 MHz, d₆- DMSO): δ 9.49 (s, 1H), 9.45 (s, 1H), 8.52(d, J = 5 Hz, 1H), 8.24 (d, J = 8.4 Hz, 2H), 7.98 (d, J = 8.4 Hz, 2H),7.65 (d, J = 9 Hz, 2H), 7.39 (d, J = 5 Hz, 1H), 6.92 (d, J = 9 Hz, 2H),3.74 (m, 4H), 3.04 (m, 4H), 1.58 (m, 2H), 1.31 (m, 2H). m/z 441.1 [M +H]⁺ N-(1- cyanocyclopropyl)- 4-(2-(4- morpholinophenyl amino)pyrimidin-4-yl)benzamide 79

484.22 ¹H NMR (300 MHz, CDCl₃): δ 8.49 (1H, d, J = 5.5 Hz), 8.17 (2H, d,J = 7.8 Hz), 7.89 (2H, d, J = 8.2 Hz), 7.75 (1H, d, J = 2.6 Hz), 7.65(1H, dd, J = 8.7, 2.3 Hz), 7.23 (1H, brs), 7.15 (1H, d, J = 5.5 Hz),7.12 (1H, d, J = 8.5 Hz), 6.65 (1H, brs), 6.08- 5.95 (1H, m), 5.37-5.30(1H, m), 5.23-5.19 (1H, m), 4.65 (2H, s), 4.42 (2H, d, J = 6.1 Hz), 4.12(2H, d, J = 5.3 Hz), 3.84 (4H, t, J = 4.4 Hz), 2.92 m/z 485.1 [M + H]⁺4-(2-(3- (allyloxymethyl)- 4- morpholinophenyl amino)pyrimidin- 4-yl)-N-(cyanomethyl) benzamide (4H, t, J = 4.6 Hz). 80

456.23 ¹H NMR (300 MHz, d₆- DMSO): δ 9.54 (s, 1H), 9.32 (m, 1H), 8.54(d, J = 5.6 Hz, 1H), 8.26 (d, J = 8.0 Hz, 2H), 8.02 (d, J = 8.0 Hz, 2H),7.68 (d, J = 8.8 Hz, 2H), 7.41 (d, J = 5.2 Hz, 1H), 6.92 (d, J = 8.8 Hz,2H), 4.31 (m, 3H), 2.69 (m, 2H), 2.32 (m, 2H), 2.14 (m, 2H), 1.93 (m,2H), 1.58 (m, m/z 457.2 [M + H]⁺ N-(cyanomethyl)- 4-(2-(4-(1-ethylpiperidin-4- yloxy)phenylamino) pyrimidin-4- yl)benzamide 2H), 1.00(m, 3H). 81

482.15 ¹H NMR (300 MHz, d₆- DMSO): δ 9.74 (1H, s), 8.57 (1H, d, J = 5.0Hz), 8.24 (2H, d, J = 8.7 Hz), 7.75 (1H, dd, J = 2.2, 15.5 Hz), 7.63(2H, d, J = 8.7 Hz), 7.52 (1H, brdd, J = 2.0, 8.7 Hz), 7.43 (1H, d, J =5.0 Hz), 7.02 (1H, dd, J = 8.7, 9.1 Hz), 4.96 (2H, s), 3.73 (4H, m),3.21 (3H, s), 2.94 (4H, m). m/z 483.0 [M + H]⁺ N-(cyanomethyl)-N-(4-(2-(3-fluoro- 4- morpholinophenyl amino)pyrimidin- 4-yl)phenyl)methane sulfonamide 82

489.16 ¹H NMR (300 MHz, d₆- DMSO): δ 9.88 (1H, s), 8.60 (1H, d, J = 5.5Hz), 8.23 (2H, d, J = 8.7 Hz), 8.17 (1H, d, J = 2.7 Hz), 8.02 (1H, dd, J= 2.7, 9.1 Hz), 7.63 (2H, d, J = 8.7 Hz), 7.48 (1H, d, J = 5.5 Hz), 7.21(1H, d, J = 9.1 Hz), 4.96 (2H, s), 3.75 (4H, m), 3.20 (3H, s), 3.06 (4H,m). m/z 490.0 [M + H]⁺ N-(4-(2-(3-cyano- 4- morpholinophenylamino)pyrimidin- 4-yl)phenyl)-N- (cyanomethyl) methanesulfonamide 83

365.14 ¹H NMR (CDCl₃/CD₃OD, 300 MHz): δ 8.47 (d, J = 5.1 Hz, 1H), 7.98(d, J = 8.4 Hz, 1H), 7.74 (d, J = 1.5 Hz, 1H), 7.57 (dd, 1.5, J = 8.4Hz, 1H), 7.44 (dd, J = 2.4, 2.1 Hz, 1H), 7.19 (dd, J = 7.8, 7.8 Hz, 1H),7.15 (d, J = 5.4 Hz, 1H), 7.05 (ddd, J = 0.9, 2.1, 8.1 Hz, 1H), 6.56(ddd, J = 0.9, 2.4, 8.1Hz, 1H), 4.36 (t, J = 6.7 Hz, 2H), 1.86 (m, 2H),1.07 (t, J = 5.7 Hz, 3H). m/z 366.3 [M + H]⁺ propyl 2-hydroxy- 4-(2-(3-hydroxyphenyl- amino)pyrimidin-4- yl)benzoate 84

393.15 ¹H NMR (300 MHz, d₆- acetone): δ 8.92 (d, J = 2.1 Hz, 1H), 8.57(d, J = 2.1 Hz, 1H), 8.4 (d, J = 5.3 Hz, 1H), 8.17 (dd, J = 8.9, 2.2 Hz,1H), 8.09 (dd, J = 9.1, 2.8 Hz, 1H), 7.25 (d, J = 5.3 Hz, 1H), 7.18 (d,J = 8.9 Hz, 1H), 6.83 (d, J = 9.1 Hz, 1H), 3.75 (m, 4H), 3.42 (m, 4H),3.22 (bs, 2H). m/z 394.1 [M + H]⁺ 4-(4-amino-3- nitrophenyl)-N-(6-morpholinopyridin- 3-yl)pyrimidin-2- amine 85

414.18 ¹H NMR (300 MHz, d₆- DMSO): δ 10.53 (s, 1H), 9.36 (s, 1H), 8.45(d, J = 5.4 Hz, 1H), 8.14 (d, J = 8.7 Hz, 2H), 7.69 (m, 4H), 7.28 (d, J= 5.4 Hz, 1H), 6.94 (d, J = 9.1 Hz, 2H), 3.96 (s, 2H), 3.74 (m, 4H),3.05 (m, 4H) m/z 415.4 [M + H]⁺ 2-cyano-N-(4-(2- (4- morpholinophenylamino)pyrimidin- 4- yl)phenyl)acetamide 86

508.15 ¹H NMR (300 MHz, d₆- DMSO): δ 17.23 (1H, s) CO₂H, 9.99 (1H, s),8.74 (1H, d, J = 2.7 Hz), 8.62 (1H, d, J = 5.0 Hz), 8.33 (2H, d, J = 8.7Hz), 8.01 (1H, dd, J = 2.7, 8.7 Hz), 7.69 (1H, d, J = 9.1 Hz), 7.63 (2H,d, J = 8.7 Hz), 7.52 (1H, d, J = 5.5 Hz), 4.97 (2H, s), 3.81 (4H, m),3.21 (3H, s), 3.06 (4H, m). m/z 509.3 [M + H]⁺ and m/z 507.4 [M − H]⁻5-(4-(4-(N- (cyanomethyl) methylsulfonamido) phenyl)pyrimidin-2-ylamino)-2- morpholinobenzoic acid 87

458.24 ¹H NMR (300 MHz, d₆- DMSO): δ 9.51 (s, 1H), 9.34 (t, J = 5.5,1H), 8.54 (d, J = 5.2 Hz, 1H), 8.26 (d, J = 8.4 Hz, 2H), 8.03 (d, J =8.4 Hz, 2H), 7.68 (d, J = 9.0 Hz, 2H), 7.41 (d, J = 5.4 Hz, 1H), 6.90(d, J = 9.0 Hz, 2H), 4.34 (d, J = 5.4 Hz, 2H), 3.98 (t, J = 6.5 Hz, 2H),2.54 (m, 6H), 1.84 (m, m/z 459.4 [M + H]⁺ N-(cyanomethyl)- 4-(2-(4-(3-(diethylamino) propoxy) phenylamino) pyrimidin-4- yl)benzamide 2H), 0.98(t, J = 7.1 Hz, 6H). 88

458.21 ¹H NMR (300 MHz, d₆- DMSO): δ 9.53 (s, 1H), 9.32 (t, J = 5.4 Hz,1H), 8.55 (d, J = 5.4 Hz, 1H), 8.26 (d, J = 8.7 Hz, 2H), 8.02 (d, J =8.7 Hz, 2H), 7.69 (d, J = 9.0 Hz, 2H), 7.42 (d, J = 5.4 Hz, 1H), 6.92(d, J = 9.3 Hz, 2H), 4.35 (d, J = 5.4 Hz, 2H), 4.06 (t, J = 5.7 Hz, 2H),3.58 (m, 4H), 2.69 (t, J = 5.8 Hz, 2H), 2.48 (m, 4H, partially obscuredby DMSO signal). m/z 459.4 [M + H]⁺ N-(cyanomethyl)- 4-(2-(4-(2-morpholinoethoxy) phenylamino) pyrimidin-4- yl)benzamide 89

462.15 ¹H NMR (300 MHz, d₆- DMSO): δ 9.52 (s, 1H), 9.32 (t, J = 5.6 Hz,1H), 8.54 (d, J = 5.1 Hz, 1H), 8.27 (d, J = 8.4 Hz, 2H), 8.02 (d, J =8.4 Hz, 2H), 7.70 (d, J = 8.7 Hz, 2H), 7.41 (d, J = 5.4 Hz, 1H), 7.02(d, J = 5.4 Hz, 2H), 4.35 (d, J = 5.1 Hz, 2H), 3.70 (m, 4H), 3.14 (m,4H). m/z 463.3 [M + H]⁺ N-(cyanomethyl)- 4-(2-{[4-(1,1- dioxo-1λ⁶,4-thiomorpholin-4- yl)phenyl]amino} pyrimidin-4- yl)benzamide 90

476.16 ¹H NMR (300 MHz, d₆- DMSO): δ 9.73 (s, 1H), 9.33 (t, J = 5.4 Hz,1H), 8.60 (d, J = 5.1 Hz, 1H), 8.29 (d, J = 8.4 Hz, 2H), 8.03 (d, J =8.7 Hz, 2H), 7.80 (d, J = 8.7 Hz, 2H), 7.48 (d, J = 5.1 Hz, 1H), 7.27(d, J = 8.7 Hz, 2H), 4.36 (d, J = 5.4 Hz, 2H), 3.62 (s, 2H), 3.10 (m,4H), 2.88 (m, 4H). m/z 477.3 [M + H]⁺ N-(cyanomethyl)- 4-[2-({4-[(1,1-dioxo-1λ⁶,4- thiomorpholin-4- yl)methyl]phenyl} amino)pyrimidin-4-yl]benzamide 91

428.20 ¹H NMR (300 MHz, d₆- DMSO): δ 9.46 (s, 1H), 8.52 (d, J = 5.1 Hz,1H), 8.23 (d, J = 8.7 Hz, 2H), 7.65 (m, 4H), 7.37 (d, J = 5.4 Hz, 1H),6.94 (d, J = 9.0 Hz, 2H), 4.56 (brs, 2H), 3.74 (m, 4H), 3.04 (m, 7H).m/z 429.3 [M + H]⁺ N-(cyanomethyl)- N-methyl-4-(2-(4- morpholinophenylamino)pyrimidin- 4-yl)benzamide 92

428.20 ¹H NMR (300 MHz, CDCl₃): δ 8.31 (s, 1H), 7.90 (d, J = 8.8 Hz,2H), 7.73 (d, J = 8.6 Hz, 2H), 7.51 (d, J = 8.6 Hz, 2H), 6.96 (brs, 1H),6.91 (d, J = 9.2 Hz, 2H), 6.56 (t, J = 5.7 Hz, 1H), 4.42 (d, J = 5.8 Hz,2H), 3.86 (t, J = 4.8 Hz, 4H), 3.11 (t, J = 4.8 Hz, 4H), 2.23 (s, 3H).m/z 429.4 [M + H]⁺ N-(cyanomethyl)- 4-(5-methyl-2-(4- morpholinophenylamino)pyrimidin- 4-yl)benzamide 93

432.17 ¹H NMR (300 MHz, CDCl₃): δ 8.35 (d, J = 3.3 Hz, 1H), 8.22 (d, J =8.1 Hz, 2H), 7.91 (d, J = 8.9 Hz, 2H), 7.51 (d, J = 9.1 Hz, 2H), 6.94(d, J = 9.0 Hz, 2H), 6.45-6.44 (m, 1H), 4.43 (d, J = 5.7 Hz, 2H), 3.88(t, J = 4.7 Hz, 4H), 3.13 (t, J = 4.8 Hz, 4H). m/z 433.3 [M + H]⁺N-(cyanomethyl)- 4-(5-fluoro-2-(4- morpholinophenyl amino)pyrimidin-4-yl)benzamide

The terms “C1-6alkyl” and “C1-4alkyl” refers to straight chain orbranched chain hydrocarbon groups having from 1 to 6 carbon atoms.Examples include ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl,tert-butyl, pentyl, neopentyl and hexyl.

The terms “C1-6alkylene” and “C1-4alkylene” are the divalent equivalentsof “C1-6alkyl” and “C1-4alkyl”.

The term “C2-4alkenyl” refers to straight chain or branched chainhydrocarbon groups having at least one double bond of either E or Zstereochemistry where applicable and 2 to 4 carbon atoms. Examplesinclude vinyl, 1-propenyl, 1-and 2-butenyl and 2-methyl-2-propenyl.

The term “aryl” refers to single, polynuclear, conjugated or fusedresidues of aromatic hydrocarbons. Examples include phenyl, biphenyl,terphenyl, quaterphenyl, naphthyl, tetrahydronaphthyl, anthracenyl,dihydroanthracenyl, benzanthracenyl, dibenxanthracenyl andphenanthrenyl.

The term “unsaturated N-containing 5 or 6-membered heterocyclyl” refersto unsaturated, cyclic hydrocarbon groups containing at least onenitrogen.

Suitable N-containing heterocyclic groups include unsaturated 5 to6-membered heteromonocyclic groups containing 1 to 4 nitrogen atoms, forexample, pyrrolyl, pyrrolinyl, imidazolyl, pyrazolyl, pyridyl,pyrimidinyl, pyrazinyl, pyridazinyl, triazolyl or tetrazolyl;unsaturated 5 or 6-membered heteromonocyclic group containing 1 to 2oxygen atoms and 1 to 3 nitrogen atoms, such as, oxazolyl, isoxazolyl oroxadiazolyl; and unsaturated 5 or 6-membered heteromonocyclic groupcontaining 1 to 2 sulphur atoms and 1 to 3 nitrogen atoms, such as,thiazolyl or thiadiazolyl.

The term “halogen” refers to fluorine, chlorine, bromine and iodine.

The term “substituted” refers to a group that is substituted with one ormore groups selected from C1-6 alkyl, C3-6 cycloalkyl, C2-6 alkenyl,C2-6 alkynyl, C1-6 alkylaryl, aryl, heterocycylyl, halo, haloC1-6alkyl,haloC3-6cycloalkyl, haloC2-6alkenyl,

haloC2-6alkynyl, haloaryl, haloheterocycylyl, hydroxy, C1-6 alkoxy,C2-6alkenyloxy, C2-6alkynyloxy, aryloxy, heterocyclyloxy, carboxy,haloC1-6alkoxy,

haloC2-6alkenyloxy, haloC2-6alkynyloxy, haloaryloxy, nitro, nitroC1-6,alkyl, nitroC2-6alkenyl, nitroaryl, nitroheterocyclyl, azido, amino,C1-6alkylamino,

C2-6alkenylamino, C2-6alkynylamino, arylamino, heterocyclamino acyl,C1-6alkylacyl, C2-6alkenylacyl, C2-6alkynylacyl, arylacyl,heterocycylylacyl, acylamino, acyloxy, aldehydo, C1-6alkylsulphonyl,arylsulphonyl, C1-6alkylsulphonylamino, arylsulphonylamino,

C1-6alkylsulphonyloxy, arylsulphonyloxy, C1-6alkylsulphenyl,C2-6alklysulphenyl, arylsulphenyl, carboalkoxy, carboaryloxy, mercapto,C1-6alkylthio, arylthio, acylthio, cyano and the like. Preferredsubstituents are selected from the group consisting of C1-4 alkyl, C3-6cycloalkyl, C2-6 alkenyl, C2-6 alkynyl, C1-6 alkylaryl, aryl,heterocycylyl, halo, haloaryl, haloheterocycylyl, hydroxy, C1-4 alkoxy,aryloxy, carboxy, amino,

C1-6alkylacyl, arylacyl, heterocycylylacyl, acylamino, acyloxy,C1-6alkylsulphenyl, arylsulphonyl and cyano.

The compounds of the invention may also be prepared as salts which arepharmaceutically acceptable, but it will be appreciated thatnon-pharmaceutically acceptable salts also fall within the scope of thepresent invention, since these are useful as intermediates in thepreparation of pharmaceutically acceptable salts. Examples ofpharmaceutically acceptable salts include salts of pharmaceuticallyacceptable cations such as sodium, potassium, lithium, calcium,magnesium, ammonium and alkylammonium; acid addition salts ofpharmaceutically acceptable inorganic acids such as hydrochloric,orthophosphoric, sulfuric, phosphoric, nitric, carbonic, boric, sulfamicand hydrobromic acids; or salts of pharmaceutically acceptable organicacids such as acetic, propionic, butyric, tartaric, maleic,hydroxymaleic, fumaric, citric, lactic, mucic, gluconic, benzoic,succinic, oxalic, phenylacetic, methanesulfonic, trihalomethanesulfonic,toluenesulfonic, benzenesulfonic, isethionic, salicylic, sulphanilic,aspartic, glutamic, edetic, stearic, palmitic, oleic, lauric,pantothenic, tannic, ascorbic, valeric and orotic acids. Salts of aminegroups may also comprise quaternary ammonium salts in which the aminonitrogen atom carries a suitable organic group such as an alkyl,alkenyl, alkynyl or aralkyl moiety.

The salts may be formed by conventional means, such as by reacting thefree base form of the compound with one or more equivalents of theappropriate acid in a solvent or medium in which the salt is insoluble,or in a solvent such as water which is removed in vacuo or by freezedrying or by exchanging the anions of an existing salt for another anionon a suitable ion exchange resin.

Where a compound possesses a chiral center the compound can be used as apurified enantiomer or diastereomer, or as a mixture of any ratio ofstereoisomers. It is however preferred that the mixture comprises atleast 70%, 80%, 90%, 95%, 97.5% or 99% of the preferred isomer, wherethe preferred isomer gives the desired level of potency and selectivity.

This invention also encompasses prodrugs of the compounds of formula I.The invention also encompasses methods of treating disorders that can betreated by the inhibition of protein kinases, such as JAK comprisingadministering drugs or prodrugs of compounds of the invention. Forexample, compounds of formula I having free amino, amido, hydroxy orcarboxylic acid groups can be converted into prodrugs. Prodrugs includecompounds wherein an amino acid residue, or a polypeptide chain of twoor more (e.g., two, three or four) amino acid residues which arecovalently joined through peptide bonds to free amino, hydroxy andcarboxylic acid groups of compounds of the invention. The amino acidresidues include the 20 naturally occurring amino acids commonlydesignated by three letter symbols and also include, 4-hydroxyproline,hydroxylysine, demosine, isodemosine, 3-methylhistidine, norvlin,beta-alanine, gamma-aminobutyric acid, citrulline, homocysteine,homoserine, ornithine and methioine sulfone. Prodrugs also includecompounds wherein carbonates, carbamates, amides and alkyl esters whichare covalently bonded to the above substituents of compounds of thepresent invention through the carbonyl carbon prodrug sidechain.Prodrugs also include phosphate derivatives of compounds (such as acids,salts of acids, or esters) joined through a phosphorus-oxygen bond to afree hydroxyl of compounds of formula I. Prodrugs may also includeN-oxides, and S-oxides of appropriate nitrogen and sulfur atoms informula I.

Process

Compounds of the general formula I are generally prepared from adichloropyrimidine.

The first step of the process typically begins with a cross-couplingreaction between a 2,4 dichloropyrimidine and a suitably functionalisedcoupling partner. Alternately the dichloropyrimidine may be converted toa diiodopyrimidine, which is then coupled with a suitably functionalisedcoupling partner. Typical coupling partners are organoboronic acids oresters (Suzuki coupling: see for example Miyaura, N. and Suzuki, ChemRev. 1995, 95 2457), organostannanes (Stille coupling: see for exampleStille, J. K., Angew. Chem., Int. Ed. Engl., 1986, 25, 508), Grignardreagents (Kumada coupling: Kumada, M.; Tamao, K.; Sumitani, K. Org.Synth. 1988, Coll. Vol. 6, 407.) or organozinc species (Negishicoupling: Negishi, E.; J. Organomet. Chem. 2002, 653, 34). The Suzukicoupling is the preferred coupling method and is typically performed ina solvent such as DME, THF, DMF, ethanol, propanol, toluene,acetonitrile or 1,4-dioxane, with or without added water, in thepresence of a base such as sodium or potassium carbonate, lithiumhydroxide, caesium carbonate, sodium hydroxide, potassium fluoride orpotassium phosphate. The reaction may be carried out at elevatedtemperatures and the palladium catalyst employed may be selected fromPd(PPh3)4, Pd(OAc)2, [PdCl2(dppf)], Pd2(dba)3/P(t-Bu)3.

The second step of the process involves a nucleophilic aromaticsubstitution reaction of the derived above with a suitably substitutedaniline. The nucleophilic aromatic substitution is typically carried outby addition of the aniline to monohalo heterocyclic intermediateobtained from the first reaction in a solvent such as ethanol,n-propanol, isopropanol, tert-butanol, dioxane, THF, DMF, toluene orxylene. The reaction is typically performed at elevated temperature inthe presence of an acid such as HCl or p-toluenesulfonic acid or in thepresence of base such as a non-nucleophilic base such as triethylamineor diisopropylethylamine, or an inorganic base such as potassiumcarbonate or sodium carbonate.

Alternatively, the aniline substituent may be introduced through atransition metal catalysed amination reaction. Typical catalysts forsuch transformations include Pd(OAc)2/P(t-Bu)3, Pd2(dba)3/BINAP andPd(OAc)2/BINAP. These reactions are typically carried out in solventssuch as toluene or dioxane, in the presence of bases such as caesiumcarbonate or sodium or potassium tert-butoxide at temperatures rangingfrom room temperature to reflux (e.g., Hartwig, J. F., Angew. Chem. Int.Ed. 1998, 37, 2046).

The anilines employed in the first step of the synthesis of thesecompounds are obtained commercially or are prepared using methods wellknown to those skilled in the art.

The products formed from either reaction step may be further derivatisedusing techniques known to those skilled in the art. Alternatively,derivatisation of the mono-halo intermediate may be undertaken prior todisplacement of the halo substituent. Those skilled in the art willappreciate that the order of the reactions described for the synthesesabove may be changed in certain circumstances and that certainfunctionalities may need to be derivatised (i.e., protected) in certaininstances for the reactions described above to proceed with reasonableyield and efficiency. The types of protecting functionality arewell-known to those skilled in the art and are described for example inGreene (Greene, T., Wuts, P. (1999) Protective Groups in OrganicSynthesis. Wiley-Interscience; 3rd edition).

The leaving group in the compound of formula II which is an intermediateused in the process of the present invention may be any suitable knowntype such as those disclosed in J. March, “Advanced Organic Chemistry:Reactions, Mechanisms and Structure” 4th Edition, pp 352-357, John Wiley& Sons, New York, 1992 which is incorporated herein by reference.Preferably, the leaving group is halogen, more preferably chlorine oriodine.

JAK Inhibition

The compounds of formula I have activity against protein kinases,particularly the JAK kinases and most particularly are active againstJAK2. A JAK2 inhibitor is any compound that selectively inhibits theactivity of JAK2. One activity of JAK2 is to phosphorylate a STATprotein. Therefore an example of an effect of a JAK2 inhibitor is todecrease the phosphorylation of one or more STAT proteins. The inhibitormay inhibit the phosphorylated form of JAK2 or the non-phosphorylatedform of JAK2.

The present invention also provides the use of kinase inhibitors such asJAK kinase inhibitors, in particular JAK2 inhibitors.

Pharmaceutical Compositions

The present invention provides pharmaceutical compositions comprising atleast one of the compounds of the formula I and a pharmaceuticallyacceptable carrier. The carrier must be “pharmaceutically acceptable”means that it is compatible with the other ingredients of thecomposition and is not deleterious to a subject. The compositions of thepresent invention may contain other therapeutic agents as describedbelow, and may be formulated, for example, by employing conventionalsolid or liquid vehicles or diluents, as well as pharmaceuticaladditives of a type appropriate to the mode of desired administration(for example, excipients, binders, preservatives, stabilizers, flavours,etc.) according to techniques such as those well known in the art ofpharmaceutical formulation (See, for example, Remington: The Science andPractice of Pharmacy, 21st Ed., 2005, Lippincott Williams & Wilkins).

The compounds of the invention may be administered by any suitablemeans, for example, orally, such as in the form of tablets, capsules,granules or powders; sublingually; buccally; parenterally, such as bysubcutaneous, intravenous, intramuscular, intra(trans)dermal, orintracisternal injection or infusion techniques (e.g., as sterileinjectable aqueous or non-aqueous solutions or suspensions); nasallysuch as by inhalation spray or insufflation; topically, such as in theform of a cream or ointment ocularly I the form of a solution orsuspension; vaginally in the form of pessaries, tampons or creams; orrectally such as in the form of suppositories; in dosage unitformulations containing non-toxic, pharmaceutically acceptable vehiclesor diluents. The compounds may, for example, be administered in a formsuitable for immediate release or extended release. Immediate release orextended release may be achieved by the use of suitable pharmaceuticalcompositions comprising the present compounds, or, particularly in thecase of extended release, by the use of devices such as subcutaneousimplants or osmotic pumps.

The pharmaceutical compositions for the administration of the compoundsof the invention may conveniently be presented in dosage unit form andmay be prepared by any of the methods well known in the art of pharmacy.These methods generally include the step of bringing the compound offormula I into association with the carrier which constitutes one ormore accessory ingredients. In general, the pharmaceutical compositionsare prepared by uniformly and intimately bringing the compound offormula I into association with a liquid carrier or a finely dividedsolid carrier or both, and then, if necessary, shaping the product intothe desired formulation. In the pharmaceutical composition the activeobject compound is included in an amount sufficient to produce thedesired effect upon the process or condition of diseases. As usedherein, the term “composition” is intended to encompass a productcomprising the specified ingredients in the specified amounts, as wellas any product which results, directly or indirectly, from combinationof the specified ingredients in the specified amounts.

The pharmaceutical compositions containing the compound of formula I maybe in a form suitable for oral use, for example, as tablets, troches,lozenges, aqueous or oily suspensions, dispersible powders or granules,emulsions, hard or soft capsules, or syrups or elixirs. Compositionsintended for oral use may be prepared according to any method known tothe art for the manufacture of pharmaceutical compositions and suchcompositions may contain one or more agents such as sweetening agents,flavouring agents, colouring agents and preserving agents, e.g., toprovide pharmaceutically stable and palatable preparations. Tabletscontain the compound of formula I in admixture with non-toxicpharmaceutically acceptable excipients which are suitable for themanufacture of tablets. These excipients may be for example, inertdiluents, such as calcium carbonate, sodium carbonate, lactose, calciumphosphate or sodium phosphate; granulating and disintegrating agents,for example, corn starch, or alginic acid; binding agents, for examplestarch, gelatin or acacia, and lubricating agents, for example magnesiumstearate, stearic acid or talc. The tablets may be uncoated or they maybe coated by known techniques to delay disintegration and absorption inthe gastrointestinal tract and thereby provide a sustained action over alonger period. For example, a time delay material such as glycerylmonostearate or glyceryl distearate may be employed. They may also becoated to form osmotic therapeutic tablets for control release.

Formulations for oral use may also be presented as hard gelatin capsuleswherein the compound of formula I is mixed with an inert solid diluent,for example, calcium carbonate, calcium phosphate or kaolin, or as softgelatin capsules wherein the compound of formula I is mixed with wateror an oil medium, for example peanut oil, liquid paraffin, or olive oil.

Aqueous suspensions contain the active materials in admixture withexcipients suitable for the manufacture of aqueous suspensions. Suchexcipients are suspending agents, for example sodiumcarboxymethylcellulose, methylcellulose, hydroxy-propylmethylcellulose,sodium alginate, polyvinyl-pyrrolidone, gum tragacanth and gum acacia;dispersing or wetting agents may be a naturally-occurring phosphatide,for example lecithin, or condensation products of an alkylene oxide withfatty acids, for example polyoxyethylene stearate, or condensationproducts of ethylene oxide with long chain aliphatic alcohols, forexample heptadecaethyleneoxycetanol, or condensation products ofethylene oxide with partial esters derived from fatty acids and ahexitol such as polyoxyethylene sorbitol monooleate, or condensationproducts of ethylene oxide with partial esters derived from fatty acidsand hexitol anhydrides, for example polyethylene sorbitan monooleate.The aqueous suspensions may also contain one or more preservatives, forexample ethyl, or n-propyl, p-hydroxybenzoate, one or more coloringagents, one or more flavoring agents, and one or more sweetening agents,such as sucrose or saccharin.

Oily suspensions may be formulated by suspending the compound of formulaI in a vegetable oil, for example arachis oil, olive oil, sesame oil orcoconut oil, or in a mineral oil such as liquid paraffin. The oilysuspensions may contain a thickening agent, for example beeswax, hardparaffin or cetyl alcohol. Sweetening agents such as those set forthabove, and flavoring agents may be added to provide a palatable oralpreparation. These compositions may be preserved by the addition of ananti-oxidant such as ascorbic acid.

Dispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water provide the compound of formula I inadmixture with a dispersing or wetting agent, suspending agent and oneor more preservatives. Suitable dispersing or wetting agents andsuspending agents are exemplified by those already mentioned above.Additional excipients, for example sweetening, flavoring and coloringagents, may also be present.

The pharmaceutical compositions of the invention may also be in the formof oil-in-water emulsions. The oily phase may be a vegetable oil, forexample olive oil or arachis oil, or a mineral oil, for example liquidparaffin or mixtures of these. Suitable emulsifying agents may benaturally-occurring gums, for example gum acacia or gum tragacanth,naturally-occurring phosphatides, for example soy bean, lecithin, andesters or partial esters derived from fatty acids and hexitolanhydrides, for example sorbitan monooleate, and condensation productsof the said partial esters with ethylene oxide, for examplepolyoxyethylene sorbitan monooleate. The emulsions may also containsweetening and flavoring agents.

Syrups and elixirs may be formulated with sweetening agents, for exampleglycerol, propylene glycol, sorbitol or sucrose. Such formulations mayalso contain a demulcent, a preservative and flavoring and coloringagents.

The pharmaceutical compositions may be in the form of a sterileinjectable aqueous or oleagenous suspension. This suspension may beformulated according to the known art using those suitable dispersing orwetting agents and suspending agents which have been mentioned above.The sterile injectable preparation may also be a sterile injectablesolution or suspension in a non-toxic parenterally-acceptable diluent orsolvent, for example as a solution in 1,3-butane diol. Among theacceptable vehicles and solvents that may be employed are water,Ringer's solution and isotonic sodium chloride solution. In addition,sterile, fixed oils are conventionally employed as a solvent orsuspending medium. For this purpose any bland fixed oil may be employedincluding synthetic mono- or diglycerides. In addition, fatty acids suchas oleic acid find use in the preparation of injectable formulations.

For administration to the respiratory tract, including intranasaladministration, the active compound may be administered by any of themethods and formulations employed in the art for administration to therespiratory tract.

Thus in general the active compound may be administered in the form of asolution or a suspension or as a dry powder.

Solutions and suspensions will generally be aqueous, for exampleprepared from water alone (for example sterile or pyrogen-free water) orwater and a physiologically acceptable co-solvent (for example ethanol,propylene glycol or polyethylene glycols such as PEG 400).

Such solutions or suspensions may additionally contain other excipientsfor example preservatives (such as benzalkonium chloride), solubilisingagents/surfactants such as polysorbates (e.g., Tween® 80, Span 80,benzalkonium chloride), buffering agents, isotonicity-adjusting agents(for example sodium chloride), absorption enhancers and viscosityenhancers. Suspensions may additionally contain suspending agents (forexample microcrystalline cellulose and carboxymethyl cellulose sodium).

Solutions or suspensions are applied directly to the nasal cavity byconventional means, for example with a dropper, pipette or spray. Theformulations may be provided in single or multidose form. In the lattercase a means of dose metering is desirably provided. In the case of adropper or pipette this may be achieved by the subject administering anappropriate, predetermined volume of the solution or suspension. In thecase of a spray this may be achieved for example by means of a meteringatomising spray pump.

Administration to the respiratory tract may also be achieved by means ofan aerosol formulation in which the compound is provided in apressurised pack with a suitable propellant, such as achlorofluorocarbon (CFC), for example dichlorodifluoromethane,trichlorofluoromethane or dichlorotetrafluoroethane, carbon dioxide orother suitable gas. The aerosol may conveniently also contain asurfactant such as lecithin. The dose of active compound may becontrolled by provision of a metered valve.

Alternatively the active compound may be provided in the form of a drypowder, for example a powder mix of the compound in a suitable powderbase such as lactose, starch, starch derivatives such ashydroxypropylmethyl cellulose and polyvinylpyrrolidine (PVP).Conveniently the powder carrier will form a gel in the nasal cavity. Thepowder composition may be presented in unit dose form, for example incapsules or cartridges of e.g., gelatin, or blister packs from which thepowder may be administered by means of an inhaler.

In formulations intended for administration to the respiratory tract,including intranasal formulations, the active compound will generallyhave a small particle size, for example of the order of 5 microns orless. Such a particle size may be obtained by means known in the art,for example by micronisation.

When desired, formulations adapted to give sustained release of theactive compound may be employed.

The active compound may be administered by oral inhalation as afree-flow powder via a “Diskhaler” (trade mark of Glaxo Group Ltd) or ameter dose aerosol inhaler.

The compounds of the present invention may also be administered in theform of suppositories for rectal administration of the drug. Thesecompositions can be prepared by mixing the drug with a suitablenon-irritating excipient which is solid at ordinary temperatures butliquid at the rectal temperature and will therefore melt in the rectumto release the drug. Such materials are cocoa butter and polyethyleneglycols.

Compositions suitable for vaginal administration may be presented aspessaries, tampons, creams, gels, pastes, foams or sprays containing inaddition to the active ingredient such carriers as are known in the artto be appropriate.

For topical use, creams, ointments, jellies, solutions or suspensions,etc., containing the compounds of the present invention are employed.(For purposes of this application, topical application shall includemouthwashes and gargles.)

For application to the eye, the active compound may be in the form of asolution or suspension in a suitable sterile aqueous or non-aqueousvehicle. Additives, for instance buffers, preservatives includingbactericidal and fungicidal agents, such as phenyl mercuric acetate ornitrate, benzalkonium chloride, or chlorohexidine and thickening agentssuch as hypromellose may also be included.

The compounds of the present invention can also be administered in theform of liposomes. As is known in the art, liposomes are generallyderived from phospholipids or other lipid substances. Liposomes areformed by mono- or multilamellar hydrated liquid crystals that aredispersed in an aqueous medium. Any non-toxic, physiologicallyacceptable and metabolisable lipid capable of forming liposomes can beused. The present compositions in liposome form can contain, in additionto a compound of the present invention, stabilisers, preservatives,excipients and the like. The preferred lipids are the phospholipids andphospha-tidyl cholines, both natural and synthetic. Methods to formliposomes are known in the art.

Efficacy of this class of compounds may be applicable to drug elutingstents. Potential applications of drug eluting stents with thesecompounds include pulmonary artery stenosis, pulmonary vein stenosis, aswell as coronary artery stenosis. Drug eluting stents may also be usedin saphenous vein grafts or arterial grafts or conduits. Drug elutingstents that release this class of compounds may also be applicable fortreating stenoses of the aorta or peripheral arteries, such as the iliacartery, the femoral artery or the popliteal artery. The compound may bebound to the drug eluting stent by any of various methods known in thefield. Examples of such methods include polymers, phosphoryl choline,and ceramics. The compound may also be impregnated into a bioabsorbablestent.

The active compounds may also be presented for use in the form ofveterinary compositions, which may be prepared, for example, by methodsthat are conventional in the art. Examples of such veterinarycompositions include those adapted for:

oral administration, external application, for example drenches (e.g.,aqueous or non-aqueous solutions or suspensions); tablets or boluses;powders, granules or pellets for admixture with feed stuffs; pastes forapplication to the tongue;

parenteral administration for example by subcutaneous, intramuscular orintravenous injection, e.g., as a sterile solution or suspension; or(when appropriate) by intramammary injection where a suspension orsolution is introduced in the udder via the teat;

topical applications, e.g., as a cream, ointment or spray applied to theskin; or

rectally or intravaginally, e.g., as a pessary, cream or foam.

The pharmaceutical composition and method of the present invention mayfurther comprise other therapeutically active compounds as noted hereinwhich are usually applied in the treatment of the above mentionedpathological conditions. Selection of the appropriate agents for use incombination therapy may be made by one of ordinary skill in the art,according to conventional pharmaceutical principles. The combination oftherapeutic agents may act synergistically to effect the treatment orprevention of the various disorders described above. Using thisapproach, one may be able to achieve therapeutic efficacy with lowerdosages of each agent, thus reducing the potential for adverse sideeffects.

Examples of other therapeutic agents include the following: endothelinreceptor antagonists (e.g., ambrisentan, bosentan, sitaxsentan), PDE-Vinhibitors (e.g., sildenafil, tadalafil, vardenafil), Calcium channelblockers (e.g., amlodipine, felodipine, varepamil, diltiazem, menthol),prostacyclin, treprostinil, iloprost, beraprost, nitric oxide, oxygen,heparin, warfarin, diuretics, digoxin, cyclosporins (e.g., cyclosporinA), CTLA4 Ig, antibodies such as ICAM 3, anti IL 2 receptor (Anti Tac),anti CD45RB, anti CD2, anti CD3 (OKT 3), anti CD4, anti CD80, anti CD86,agents blocking the interaction between CD40 and gp39, such asantibodies specific for CD40 and/or gp39 (i.e., CD154), fusion proteinsconstructed from CD40 and gp39 (CD401g and CD8gp39), inhibitors, such asnuclear translocation inhibitors, of NF kappa B function, such asdeoxyspergualin (DSG), cholesterol biosynthesis inhibitors such as HMGCoA reductase inhibitors (lovastatin and simvastatin), non-steroidalanti-inflammatory drugs (NSAIDs) such as ibuprofen, aspirin,acetaminophen, leflunomide, deoxyspergualin, cyclooxygenase inhibitorssuch as celecoxib, steroids such as prednisolone or dexamethasone, goldcompounds, beta-agonists such as salbutamol, LABA's such as salmeterol,leukotriene antagonists such as montelukast, antiproliferative agentssuch as methotrexate, FK506 (tacrolimus, Prograf), mycophenolatemofetil, cytotoxic drugs such as azathioprine, VP-16, etoposide,fludarabine, doxorubin, adriamycin, amsacrine, camptothecin, cytarabine,gemcitabine, fluorodeoxyuridine, melphalan and cyclophosphamide,antimetabolites such as methotrexate, topoisomerase inhibitors such ascamptothecin, DNA alkylators such as cisplatin, kinase inhibitors suchas sorafenib, microtubule poisons such as paclitaxel, TNF-inhibitorssuch as tenidap, anti-TNF antibodies or soluble TNF receptor, hydroxyurea and rapamycin (sirolimus or Rapamune) or derivatives thereof.

When other therapeutic agents are employed in combination with thecompounds of the present invention they may be used for example inamounts as noted in the Physician Desk Reference (PDR) or as otherwisedetermined by one of ordinary skill in the art.

Methods of Treatment

The compounds of formula I may be used in the treatment of kinaseassociated diseases including JAK kinase associated diseases such asimmunological and inflammatory diseases including organ transplants;hyperproliferative diseases including cancer and myeloproliferativediseases; viral diseases; metabolic diseases; and vascular diseases.

Generally, the term “treatment” means affecting a subject, tissue orcell to obtain a desired pharmacological and/or physiological effect andinclude: (a) preventing the disease from occurring in a subject that maybe predisposed to the disease, but has not yet been diagnosed as havingit; (b) inhibiting the disease, i.e., arresting its development; or (c)relieving or ameliorating the effects of the disease, i.e., causeregression of the effects of the disease.

The term “subject” refers to any animal having a disease which requirestreatment with the compound of formula I.

In addition to primates, such as humans, a variety of other mammals canbe treated using the compounds, compositions and methods of the presentinvention. For instance, mammals including, but not limited to, cows,sheep, goats, horses, dogs, cats, guinea pigs, rats or other bovine,ovine, equine, canine, feline, rodent or murine species can be treated.However, the invention can also be practiced in other species, such asavian species (e.g., chickens).

The term “administering” should be understood to mean providing acompound of the invention to a subject in need of treatment.

The term “kinase associated diseases” refers to a disorder or disordersthat directly or indirectly result from or are aggravated by aberrantkinase activity, in particular JAK activity and/or which are alleviatedby inhibition of one or more of these kinase enzymes.

In a preferred embodiment the kinase associated disease state involvesone or more of the JAK kinases, JAK1, JAK2, JAK3 or TYK2. In aparticularly preferred embodiment, the disease involves JAK2 kinase.Such diseases include, but are not limited to, those listed in the Tablebelow.

Activation of the JAK/STAT pathway in various pathologies Cell TypesCytokines JAK Kinase Disease Type Involved involved InvolvedCharacteristics Atopy Allergic Asthma, Atopic Mast Cells, IL-4, IL-5,IL- JAK1, T-cell activation of B- Dermatitis (Eczema), Eosinophils, T-6, IL-7, IL-13 JAK2, cells followed by IgE Allergic Rhinitis Cells,B-Cells JAK3, mediated activation of Tyk2 resident Mast cells andEosinophils CMI Allergic Contact T-cells, B-cells, IL-2, IL-4, IL- JAKEB cell and/or T_(DH) cell Dermatitis, macrophages, 5, IL-6, IL-10, JAK2,activation hypersensitivity neutrophils IFNγ, TNF, IL- JAK3, Macrophage/pneumonitis 7, IL-13, Tyk2 granulocyte activation AutoImmune DiseasesMultiple sclerosis, B-Cells, T-cells, IL-2, IL-4, IL- JAK1, CytokineProduction Glomerulonephritis, monocytes, 5, IL-6, IL-7, JAK2, (e.g.,TNFα/β, IL-1, Systemic Lupus, Macrophages, Il-10, IL-13, JAK3, CSF-1,GM-CSF), T- Erythematosus (SLE), Neutrophils, IFNγ, TNF, Tyk2 cellActivation, B-cell Rheumatoid Arthritis, Mast Cells, GM-CSF; activation,JAK/STAT Juvenile Arthritis, Eosinophils, G-CSF activation Sjögren'sSyndrome, Scleroderma Polymyositis, Ankylosing Spondylitis, PsoriaticArthritis Transplantation Allograft Rejection T cells, B cells, IL-2,IL-4, IL- JAKE Macrophage/T cell GvHD macrophages 5, IL-7, IL-13, JAK2,mediated necrosis, Tc TNF JAK3 cell mediated apoptosis, and B- cell/Igmediated opsonization/necrosis of foreign graft Viral Diseases EpsteinBarr Virus (EBV) Lymphocytes Viral JAK1, JAK/STAT Mediation Hepatitis BHepatocytes Cytokines, JAK2, Hepatitis C Hepatocytes IL-2 JAK3 HIVLymphocytes HTLV 1 Lymphocytes Varicella-Zoster Virus Fibroblasts (VZV)Human Papilloma Virus Epithelial cells (HPV) Hyperproliferativediseases-cancer Leukemia Leucocytes Various JAK1, Cytokine production,Lymphoma Lymphocytes Autocrine JAK2, JAK/STAT Activation MultipleMyeloma various cytokines, JAK3 prostate cancer various Intrinsic breastcancer various Activation hodgkins lympohoma various B-cell chronicvarious lymphocytic leukemia lung cancer various hepatoma variousmetastatic melanoma various glioma various Myeloproliferative DiseasesPolycythemia vera (PV), Hematopoietic Interleukin-3, JAK2 JAK/STATactivation primary myelofibrosis, erythropoietin, mutationthrombocythemia, thrombopoietin essential thrombocythemia (ET),idiopathic myelofibrosis, chronic myelogenous leukemia, systemicmastocystosis (SM), chronic neutrophilic leukemia (CNL), myelodisplasticsyndrome (MDS), systemic mast cell disease (SMCD) Vascular DiseaseHypertension, Endothelial IL6, JAK1, JAK/STAT activation Hypertrophy,Heart cells, smooth angiotensin II, JAK2, Failure, Ischemia, musclecells LIF, TYK2 Pulmonary arterial including TNFalpha, hypertensionpulmonary serotonin, artery smooth caveolin1 muscle cells, cardiacmyocytes, fibroblasts, endothelial cells Metabolic disease Obesity,metabolic Adipocytes, Leptin JAK2 JAK/STAT activation syndrome pituitarycells, neurons, monocytes

The term “immunological and inflammatory disease” refers to animmunological, inflammatory or autoimmune disease, including but notlimited to rheumatoid arthritis, polyarthritis, rheumatoid spondylitis,osteoarthritis, gout, asthma, bronchitis, allergic rhinitis, chronicobstructive pulmonary disease, cystic fibrosis, inflammatory boweldisease, irritable bowel syndrome, mucous colitis, ulcerative colitis,diabrotic colitis, Crohn's disease, autoimmune thyroid disorders,gastritis, esophagitis, hepatitis, pancreatitis, nephritis, psoriasis,eczema, acne vulgaris, dermatitis, hives, multiple sclerosis,Alzheimer's disease, Motor Neurone Disease (Lou Gehrig's disease),Paget's disease, sepsis, conjunctivitis, neranl catarrh, chronicarthrorheumatism, systemic inflammatory response syndrome (SIRS),polymyositis, dermatomyositis (DM), Polaritis nodoa (PN), mixedconnective tissue disorder (MCTD), Sjögren's syndrome, Crouzon syndrome,achondroplasia, systemic lupus erythematosus, scleroderma, vasculitis,thanatophoric dysplasia, insulin resistance, Type I diabetes andcomplications from diabetes and metabolic syndrome.

The term “hyperproliferative diseases” includes cancer andmyeloproliferative disease states such as cellular-proliferative diseasestates, including but not limited to: Cardiac: sarcoma (angiosarcoma,fibrosarcoma, rhabdomyosarcoma, liposarcoma), myxoma, rhabdomyoma,fibroma, lipoma and teratoma; Lung: bronchogenic carcinoma (squamouscell, undifferentiated small cell, undifferentiated large cell,adenocarcinoma), alveolar (bronchiolar) carcinoma, bronchial adenoma,sarcoma, lymphoma, chondromatous hanlartoma, inesothelioma;Gastrointestinal: esophagus (squamous cell carcinoma, adenocarcinoma,leiomyosarcoma, lymphoma), stomach (carcinoma, lymphoma,leiomyosarcoma), pancreas (ductal adenocarcinoma, insulinorna,glucagonoma, gastrinoma, carcinoid tumors, vipoma), small bowel(adenocarcinoma, lymphoma, carcinoid tumors, Karposi's sarcoma,leiomyoma, hemangioma, lipoma, neurofibroma, fibroma), large bowel(adenocarcinoma, tubular adenoma, villous adenoma, hamartoma,leiomyoma); Genitourinary tract: kidney (adenocarcinoma, Wilms tumor[nephroblastoma], lymphoma, leukemia), bladder and urethra (squamouscell carcinoma, transitional cell carcinoma, adenocarcinoma), prostrate(adenocarcinoma, sarcoma), testis (seminoma, teratoma, embryonalcarcinoma, teratocarcinoma, choriocarcinoma, sarcoma, interstitial cellcarcinoma, fibroma, fibroadenoma, adenomatoid tumors, lipoma); Liver:hepatoma (hepatocellular carcinoma), cholangiocarcinoma, hepatoblastoma,angiosarcoma, hepatocellular adenoma, hemangioma; Bone: osteogenicsarcoma (osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma,chondrosarcoma, Ewing's sarcoma, malignant lymphoma (reticulum cellsarcoma), multiple myeloma, malignant giant cell tumor chordoma,osteochronfrorna (osteocartilaginous exostoses), benign chondroma,chondroblastoma, chondromyxofibroma, osteoid osteoma and giant celltumors; Nervous system: skull (osteoma, hemangioma, granuloma, xanthoma,osteitis defornians), meninges (meningioma, meningiosarcoma,gliomatosis), brain (astrocytoma, medulloblastoma, glioma, ependymoma,germinoma [pinealoma], glioblastoma multiform, oligodendroglioma,schwannoma, retinoblastoma, congenital tumors), spinal cordneurofibroma, meningioma, glioma, sarcoma); Gynecological: uterus(endometrial carcinoma), cervix (cervical carcinoma, pre-tumor cervicaldysplasia), ovaries (ovarian carcinoma [serous cystadenocarcinoma,mucinous cystadenocarcinoma, unclassified carcinoma], granulosa-thecalcell tumors, SertoliLeydig cell tumors, dysgerminoma, malignantteratoma), vulva (squamous cell carcinoma, intraepithelial carcinoma,adenocarcinoma, fibrosarcoma, melanoma), vagina (clear cell carcinoma,squamous cell carcinoma, botryoid sarcoma [embryonal rhabdomyosarcoma]),fallopian tubes (carcinoma); Hematologic: blood (myeloid leukemia [acuteand chronic], acute lymphoblastic leukemia, chronic lymphocyticleukemia, multiple myeloma, myelodysplastic syndrome), Hodgkin'sdisease, non-Hodgkin's lymphoma [malignant lymphoma; Skin: malignantmelanoma, basal cell carcinoma, squamous cell carcinoma, Karposi'ssarcoma, moles dysplastic nevi, lipoma, angioma, dermatofibroma,keloids, psoriasis; Adrenal glands: neuroblastoma; andMyleoproliferative diseases such as polycythemia vera (PV), primarymyelofibrosis, thrombocythemia, essential thrombocythemia (ET), agnoneicmyeloid metaplasia (AMM), also referred to as idiopathic myelofibrosis(IMF), chronic myelogenous leukemia (CML), systemic mastocystosis (SM),chronic neutrophilic leukemia (CNL), myelodisplastic syndrome (MDS) andsystemic mast cell disease (SMCD).

The term “vascular diseases” refers to diseases including but notlimited to cardiovascular diseases, hypertension, hypertrophy,hypercholesterolemia, hyperlipidemia, thrombotic disorders, stroke,Raynaud's phenomenon, POEMS syndrome, angina, ischemia, migraine,peripheral arterial disease, heart failure, restenosis, atherosclerosis,left ventricular hypertrophy, myocardial infarction, ischemic diseasesof heart, kidney, liver and brain, and pulmonary arterial hypertension.

Preferred diseases for JAK2 selective inhibitors include immunologicaland inflammatory diseases such as auto-immune diseases for exampleatopic dermatitis, asthma, rheumatoid arthritis, Crohn's disease,psoriasis, Crouzon syndrome, achondroplasia, systemic lupuserythematosus, scleroderma, mixed connective tissue disease, vasculitis,thanatophoric dysplasia and diabetes; hyperproliferative disorders suchas cancer for example prostate cancer, colon cancer, breast cancer,liver cancer such as hepatoma, lung cancer, head and neck cancer such asglioma, skin cancer such as metastatic melanoma, leukemia, lymphoma,multiple myeloma and myeloproliferative diseases such as polycythemiavera (PV), myelofibrosis, thrombocythemia, essential thrombocythemia(ET), agnoneic myeloid metaplasia (AMM), also referred to as idiopathicmyelofibrosis (IMF) and chronic myelogenous leukemia (CML); and vasculardiseases such as hypertension, hypertrophy, stroke, Raynaud'sphenomenon, POEMS syndrome, angina, ischemia, migraine, peripheralarterial disease, heart failure, restenosis, atherosclerosis andpulmonary arterial hypertension.

Dosages

The term “therapeutically effective amount” refers to the amount of thecompound of formula I and II that will elicit the biological or medicalresponse of a tissue, system, animal or human that is being sought bythe researcher, veterinarian, medical doctor or other clinician.

In the treatment or prevention of conditions which require kinaseinhibition an appropriate dosage level will generally be about 0.01 to500 mg per kg patient body weight per day which can be administered insingle or multiple doses. Preferably, the dosage level will be about 0.1to about 250 mg/kg per day; more preferably about 0.5 to about 100 mg/kgper day. A suitable dosage level may be about 0.01 to 250 mg/kg per day,about 0.05 to 100 mg/kg per day, or about 0.1 to 50 mg/kg per day.Within this range the dosage may be 0.05 to 0.5, 0.5 to 5 or 5 to 50mg/kg per day. For oral administration, the compositions are preferablyprovided in the form of tablets containing 1.0 to 1000 milligrams of theactive ingredient, particularly 1.0, 5.0, 10.0, 15.0, 20.0, 25.0, 50.0,75.0, 100.0, 150.0, 200.0, 250.0, 300.0, 400.0, 500.0, 600.0, 750.0,800.0, 900.0, and 1000.0 milligrams of the active ingredient. The dosagemay be selected, for example to any dose within any of these ranges, fortherapeutic efficacy and/or symptomatic adjustment of the dosage to thepatient to be treated. The compounds will preferably be administered ona regimen of 1 to 4 times per day, preferably once or twice per day.

It will be understood that the specific dose level and frequency ofdosage for any particular patient may be varied and will depend upon avariety of factors including the activity of the specific compoundemployed, the metabolic stability and length of action of that compound,the age, body weight, general health, sex, diet, mode and time ofadministration, rate of excretion, drug combination, the severity of theparticular condition, and the host undergoing therapy.

In order to exemplify the nature of the present invention such that itmay be more clearly understood, the following non-limiting examples areprovided.

EXAMPLES

Compound Synthesis

The compounds of the invention may be prepared by methods well known tothose skilled in the art, and as described in the synthetic andexperimental procedures shown below for selected compounds.

Definitions

-   -   PyBOP benzotriazole-1-yloxytripyrrolidinophosphonium        hexafluorophosphate    -   DMF N,N-dimethylformamide    -   DMAP 4-Dimethylaminopyridine    -   DCM dichloromethone    -   NMP 1-methyl-2-pyrorrolidinone    -   n-PrOH n-propanol    -   ACN acetonitrile    -   EDC.HCl 1-ethyl-3-(dimethylaminopropyl)carbodiimide        hydrochloride    -   HOBT N-hydroxybenzotriazole    -   TEA triethylamine    -   DIPEA diisopropylethylamine    -   p-TsOH p-toluene sulfonic acid    -   HATU o-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium        hexafluorophosphate

Example 1—Synthesis of Compound 3

A mixture of 4-ethoxycarbonylphenyl boronic acid (23.11 g, 119 mmol),2,4-dichloropyrimidine (16.90 g, 113 mmol), toluene (230 mL) and aqueoussodium carbonate (2 M, 56 mL) was stirred vigorously and nitrogen wasbubbled through the suspension for 15 minutes.Tetrakis(triphenylphosphine)palladium[0] (2.61 g, 2.26 mmol) was added.Nitrogen was bubbled through for another 10 min., the mixture was heatedto 100° C., then at 75° C. overnight. The mixture was cooled, dilutedwith ethyl acetate (200 mL), water (100 mL) was added and the layerswere separated. The aqueous layer was extracted with ethyl acetate (100ml) and the two organic extracts were combined. The organics were washedwith brine, filtered through sodium sulfate, concentrated, and theresultant solid was triturated with methanol (100 mL) and filtered. Thesolids were washed with methanol (2×30 mL) and air dried. This materialwas dissolved in acetonitrile (150 mL) and dichloromethane (200 mL),stirred with MP.TMT Pd-scavenging resin (Agronaut part number 800471)(7.5 g) over 2 days. The solution was filtered, the solids were washedwith dichloromethane (2×100 mL), and the filtrate concentrated to giveethyl 4-(2-chloropyrimidin-4-yl)benzoate as an off-white solid (17.73 g,60%)—additional washing with dichloromethane yielded a further 1.38 gand 0.5 g of product. ¹H NMR (300 MHz, d₆-DMSO) δ 8.89 (1H, d, J=5.0Hz); 8.32 (2H, d, J=8.7 Hz); 8.22 (1H, d, J=5.5 Hz); 8.12 (2H, d, J=8.7Hz); 4.35 (2H, q, J=7.1 Hz); 1.34 (3H, t, J=7.1 Hz); LC-ESI-MS (methodB): rt 7.3 min.; m/z 263.0/265.0 [M+H]⁺.

A mixture of ethyl 4-(2-chloropyrimidin-4-yl)benzoate (26.15 g, 99.7mmol) and 4-morpholinoaniline (23.10 g, 129.6 mmol) was suspended in1,4-dioxane (250 mL). p-Toluenesulfonic acid monohydrate (17.07 g, 89.73mmol) was added. The mixture was heated at reflux for 40 h., cooled toambient temperature, concentrated then the residue was partitionedbetween ethyl acetate and 1:1 saturated sodium bicarbonate/water (1 Ltotal). The organic phase was washed with water (2×100 mL) andconcentrated. The aqueous phase was extracted with dichloromethane(3×200 mL). The material which precipitated during this workup wascollected by filtration and set aside. The liquid organics werecombined, concentrated, triturated with methanol (200 mL) and filteredto yield additional yellow solid. The solids were combined, suspended inmethanol (500 mL), allowed to stand overnight then sonicated andfiltered. The solids were washed with methanol (2×50 mL) to give, afterdrying, ethyl 4-(2-(4-morphonlinophenylamino)pyrimidin-4-yl)benzoate(35.39 g, 88%). ¹H NMR (300 MHz, d₆-DMSO) δ 9.49 (1H, s); 8.54 (1H, d,J=5.0 Hz); 8.27 (2H, d, J=8.7 Hz); 8.10 (2H, d, J=8.7 Hz), 7.66 (2H, d,J=9.1 Hz); 7.38 (1H, d, J=5.0 Hz); 6.93 (2H, d, J=8.7 Hz); 4.35 (2H, q,J=6.9 Hz), 3.73 (4H, m); 3.04 (4H, m); 1.34 (3H, t, J=6.9 Hz); LC-ESI-MS(method B): rt 7.5 min.; m/z 404.1 [M+H]⁺.

A solution of ethyl4-(2-(4-morpholinophenylamino)pyrimidin-4-yl)benzoate (35.39 g, 87.6mmol) in 3:1 methanol/tetrahydrofuran (350 mL) was treated with lithiumhydroxide (4.41 g, 183.9 mmol) in water (90 mL). The mixture was heatedat reflux for 2 h., cooled, concentrated and acidified with hydrochloricacid (2M, 92.5 mL, 185 mmol). The dark precipitate was filtered, washedwith water, and dried under vacuum. The solid was ground to a powderwith a mortar and pestle, triturated with methanol (500 mL) thenfiltered again to yield4-(2-(4-morpholinophenylamino)pyrimidin-4-yl)benzoic acid as a muddysolid. This material was washed with ether, air dried overnight, andground to a fine powder with mortar and pestle. On the basis of massrecovery (34.49 g) the yield was assumed to be quantitative. ¹H NMR (300MHz, d₆-DMSO) δ 9.47 (1H, s); 8.53 (1H, d, J=5.2 Hz); 8.24 (2H, d, J=8.5Hz); 8.08 (2H, d, J=8.8 Hz), 7.66 (2H, d, J=9.1 Hz); 7.37 (1H, d, J=5.2Hz); 6.93 (2H, d, J=9.1 Hz); 3.73 (4H, m); 3.04 (4H, m). LC-ESI-MS(method C): rt 7.3 min.; m/z 377.1 [M+H]⁺.

To a suspension of 4-(2-(4-morpholinophenylamino)pyrimidin-4-yl)benzoicacid (theoretically 32.59 g, 86.6 mmol) in DMF (400 mL) was addedtriethylamine (72.4 mL, 519.6 mmol, 6 eq.) The mixture was sonicated toensure dissolution. Aminoacetonitrile hydrochloride (16.02 g, 173.2mmol) was added followed by N-hydroxybenzotriazole (anhydrous, 14.04 g,103.8 mmol) and 1-ethyl-3-(dimethylaminopropyl)carbodiimidehydrochloride (19.92 g, 103.8 mmol). The suspension was stirredvigorously overnight. The solvent was evaporated under reduced pressure,the residue was diluted with 5% sodium bicarbonate (400 mL) and water(300 mL), giving a yellow solid, which was broken up and filtered. Thesolids were washed several times with 100 mL portions of water,triturated with hot methanol/dichloromethane (500 mL, 1:1), concentratedto a volume of approximately 300 mL), cooled and filtered. The solidswere washed with cold methanol (3×100 mL), ether (200 mL) and hexane(200 mL) prior to drying to afford Compound 3 (31.69 g, 88%). M.p.238-243° C. Microanalysis: Found C, 66.52; H, 5.41; N, 20.21.C₂₃H₂₆N₆O₁₀S₂ requires C, 66.65; H, 5.35; N, 20.28%. ¹³C NMR (75.5 MHz,d₆-DMSO) δ 166.04, 162.34, 160.26, 159.14, 146.14, 139.87, 134.44,132.73, 127.80, 126.84, 120.29, 117.49, 115.50, 107.51, 66.06, 49.16,27.68.

Example 2—Synthesis of Compound 47

To a solution of 2,4-dichloro-5-methylpyrimidine (244 mg, 1.5 mmol) andmethyl 2-methoxy-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoate(210 mg, 1.0 mmol) in toluene (3 mL) were added n-propanol (1 mL),aqueous sodium bicarbonate (2 M, 1.5 μL) andtetrakis(triphenylphosphine)palladium[0] (116 mg, 0.1 mmol). Thereaction was heated at 110° C. for 40 h, then partitioned between ethylacetate and saturated aqueous sodium bicarbonate. The aqueous layer wasextracted twice further with ethyl acetate and the combined organicfractions were washed with water, brine then dried (sodium sulfate),filtered and concentrated. Silica gel chromatography using 30-60% ethylacetate/petroleum spirit as eluent provided methyl4-(2-chloro-5-methylpyrimidin-4-yl)-2-methoxybenzoate as a cream solid(165 mg, 56%); LC-ESI-MS (method B): rt 6.2 min.; m/z 293.3/295.3[M+H]⁺.

To a solution of methyl4-(2-chloro-5-methylpyrimidin-4-yl)-2-methoxybenzoate (165 mg, 0.56mmol) in 1,4-dioxane (5 mL) was added 4-morpholinoaniline (96 mg, 0.54mmol) and p-toluenesulfonic acid monohydrate (97 mg, 0.51 mmol). Thereaction was heated at reflux for 40 h, cooled to room temperature andpartitioned between ethyl acetate and saturated aqueous sodiumbicarbonate. The aqueous layer was extracted twice more with ethylacetate and the combined organic fractions were washed twice with 5%aqueous citric acid, water, brine then dried (sodium sulfate) filteredand concentrated to afford the crude product. Trituration with methanolprovided methyl4-(2-(4-morpholinophenylamino)-5-methylpyrimidin-4-yl)-2-methoxybenzoateas a yellow solid (77 mg, 32%); ¹H NMR (300 MHz, d₆-DMSO) δ 9.36 (s,1H), 8.38 (s, 1H), 7.76 (d, J=8.1 Hz, 1H), 7.62 (d, J=9.0 Hz, 2H), 7.38(s, 1H), 7.27 (d, J=8.1 Hz, 1H), 6.88 (d, J=9.0 Hz, 2H), 3.89 (s, 3H),3.82 (s, 3H), 3.72 (m, 4H), 3.01 (m, 4H), 2.12 (s, 3H); LC-ESI-MS(method B): rt 6.7 min.; m/z 435.3 [M+H]⁺.

To a solution of methyl4-(2-(4-morpholinophenylamino)-5-methylpyrimidin-4-yl)-2-methoxybenzoate(70 mg, 0.16 mmol) in 1,4-dioxane (5 mL) was added aqueous sodiumhydroxide (5 M, 5 mL). The reaction was heated at reflux overnight thencooled to room temperature. The yellow solid which precipitated wascollected by filtration and washed with water to afford the sodium saltof Compound 40 in quantitative yield.

The sodium salt of4-(2-(4-morpholinophenylamino)-5-methylpyrimidin-4-yl)-2-methoxybenzoicacid (Compound 40) (0.16 mmol), was acidified by suspending in ethylacetate and partitioning against 5% aqueous citric acid. Furtherextraction with ethyl acetate followed by evaporation of the solventthen furnished the free acid which was suspended in dichloromethane (3mL). To this solution was added triethylamine (111 μL, 0.8 mmol),1-ethyl-3-(dimethylaminopropyl)carbodiimide hydrochloride (58 mg, 0.3mmol), aminoacetonitrile hydrochloride (61 mg, 0.4 mmol) and a catalyticamount of N,N-dimethyl aminopyridine. N,N-Dimethyl formamide (2 mL) wasadded to aid solubility and the reaction was stirred for 64 h. Thereaction was incomplete by TLC analysis so0-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (76 mg, 0.2 mmol) was added and the reaction stirredfor a further 24 h before being partitioned between dichloromethane andsaturated aqueous sodium bicarbonate. The aqueous layer was extractedtwice further with dichloromethane and the combined organics washed withwater, brine then dried (sodium sulfate) filtered and concentrated toafford the crude product. Silica gel chromatography using 0-3%methanol/ethyl acetate as the eluent afforded, as a green/yellow solid,Compound 47 (13.2 mg, 18%).

Example 3—Synthesis of Compound 90

To a suspension of 4-carboxyphenylboronic acid (5.0 g, 30 mmol) in DMF(5 mL) and dichloromethane (200 mL) at 0° C. was added oxalylchloride(5.9 mL, 66 mmol) dropwise. When gas evolution slowed, the ice bath wasremoved and the reaction allowed to warm to room temperature over 30min. The reaction was then heated at 40° C. for three hours by whichtime all solids had dissolved. The dichloromethane was removed bydistillation and the DMF solution cooled to 0° C. A solution ofaminoacetonitrile hydrochloride (3.05 g, 33 mmol) in DMF (80 mL) andDIPEA (13 mL, 75 mmol) was then added dropwise. After the addition wascomplete the ice bath was removed and the solution allowed to stir atroom temperature for 16 h. Most of the DMF was then removed in vacuo andthe reaction was partitioned between ethyl acetate and 2 M aqueoushydrochloric acid. The aqueous layer was extracted twice further withethyl acetate and the combined organic fractions dried (Na₂SO₄) filteredand concentrated under reduced pressure to afford4-(cyanomethylcarbamoyl)phenylboronic acid as a waxy pale yellow solid(5.34 g, 87%). ¹H NMR (300 MHz, d₆-DMSO): 9.18 (br. t, J=5.1 Hz, 1H),7.8-7.9 (m, 4H), 4.31 (d, J=5.4 Hz, 2H); LC-ESI-MS (method B): rt 0.9min.; m/z 203.3 [M−H]⁻.

To a solution of 2,4-dichloropyrimidine (3.2 g, 0.22 mmol) and4-(cyanomethylcarbamoyl)phenylboronic acid (3.0 g, 15 mmol) in toluene(146 mL) were added n-propanol (44 mL), aqueous sodium bicarbonate (2M,22 mL) and tetrakis(triphenylphosphine) palladium[0] (850 mg, 0.7 mmol).The reaction was heated at 90° C. for 24 h, then partitioned betweenethyl acetate and water. The aqueous layer was extracted twice furtherwith ethyl acetate and the combined organic fractions washed with brine,dried (Na₂SO₄) filtered and concentrated. Silica gel chromatographyusing 30-70% ethyl acetate/petroleum spirit as eluent provided4-(2-chloropyrimidin-4-yl)-N-(cyanomethyl)benzamide as a pale yellowwaxy solid (1.35 g, 33%). ¹H NMR (300 MHz, d₆-DMSO) δ 9.40 (t, J=5.4 Hz,1H), 8.88 (d, J=5.2 Hz, 1H), 8.32 (d, J=8.7 Hz, 2H), 8.23 (d, J=5.1 Hz,1H), 8.05 (d, J=8.7 Hz, 2H), 4.36 (d, J=5.4 Hz, 2H); LC-ESI-MS (methodB): rt 5.3 min.; m/z 273.2/275.2 [M+H]⁺.

A Schlenck flask was dried with a heat gun under vacuum for two minutesand then backfilled at room temperature with nitrogen.Tris(dibenzylideneacetone)dipalladium (9 mg, 0.01 mmol),(2-biphenylyl)di-tert-butylphosphine (5.7 mg, 0.02 mmol), potassiumphosphate (56 mg, 0.27 mmol),4-(2-chloropyrimidin-4-yl)-N-(cyanomethyl)benzamide (52 mg, 0.19 mmol)and 4-[(1,1-dioxidothiomorpholin-4-yl)methyl]aniline (40 mg, 0.17 mmol)were added and mixed together in the flask under a constant flow ofnitrogen. The flask was sealed, evacuated under high vacuum and thenbackfilled with nitrogen. The operation was repeated twice.1,2-dimethoxyethane (1.9 mL) was added through the rubber septum. Theflask was sealed and vigorous stirring was initiated. The mixture wasthen frozen with liquid nitrogen, degassed under high vacuum and thenbackfilled with nitrogen (the operation was repeated twice). The sealedflask was then heated to 100° C. overnight. A small amount oftris(dibenzylideneacetone) dipalladium and(2-biphenylyl)di-tert-butylphosphine was added, the mixture was frozen,degassed under high vacuum and backfilled with nitrogen before beingheated at 100° C. for a further 16 h. Ethyl acetate was added and themixture filtered through a sintered funnel. The filtrate was thenconcentrated and ethyl acetate added. The resulting mixture was thenwashed with a solution of citric acid (2%) and a saturated solution ofsodium chloride. The organic layer was dried (sodium sulfate), filteredand evaporated to give the crude product which was purified by columnchromatography using petroleum spirit/ethyl acetate (1/4) to give aresidue which was triturated with methanol to give Compound 90 (5.5 mg,7%).

Example 4—Synthesis of Compound 73

A round bottomed flask was charged with4-methanesulfonylaminophenylboronic acid (4.30 g, 20 mmol) and2,4-dichloropyrimidine (5.97 g, 40 mmol, 2 eq.), toluene (75 mL),n-propanol (25 mL) and aqueous sodium carbonate solution (2M, 18 mL, 1.8eq.). The reaction mixture was evacuated and backfilled with nitrogenthree times before adding tetrakis(triphenylphosphine) palladium (0)catalyst (1.02 g, 4.4 mol %). The reaction mixture was again evacuatedand backfilled with nitrogen three times before being heated at 100° C.under a nitrogen atmosphere for 66 hours. The reaction mixture wascooled and stirred at room temperature for several hours during whichtime the product precipitated from the reaction mixture. The fine yellowsolid (3.45 g, 61% yield) was collected by vacuum filtration, washedwith methanol and dried under high vacuum. ¹H NMR and LC MS dataconfirmed this to be the desiredN-(4-(2-chloropyrimidin-4-yl)phenyl)methanesulfonamide. ¹H NMR (300 MHz,d₆-DMSO) δ 10.26 (1H, brs); 8.75 (1H, d, J=5.5 Hz); 8.17 (2H, d, J=9.1Hz); 8.05 (1H, d, J=5.5 Hz); 7.35 (2H, d, J=8.7 Hz); 3.10 (3H, s).LC-ESI-MS (method B): rt 5.5 min.; m/z 284.2/286.1 [M+H]⁺.

N-(4-(2-chloropyrimidin-4-yl)phenyl)methanesulfonamide (750 mg 2.64mmol) and potassium carbonate (730 mg, 2 eq.) were placed in a roundbottomed flask and suspended in acetone (50 mL). The mixture was stirredfor several minutes before adding bromoacetonitrile (368 μL, 2 eq.). Thereaction mixture was stirred at room temperature for 48 h. The crudereaction mixture was concentrated in vacuo and the residue taken up inethyl acetate (200 mL) and washed with water (2×100 mL), brine (100 mL)and then dried (sodium sulfate). The organic phase was concentrated invacuo to giveN-(4-(2-chloropyrimidin-4-yl)phenyl)-N-(cyanomethyl)methanesulfonamide(762 mg, 89% yield) as a fawn solid. ¹H NMR (300 MHz, d₆-DMSO) δ 8.86(1H, d, J=5.0 Hz); 8.28 (2H, d, J=8.7 Hz); 8.18 (1H, d, J=5.5 Hz); 7.66(2H, d, J=8.7 Hz); 4.97 (2H, s); 3.22 (3H, s). LC-ESI-MS (method B): rt5.9 min.; m/z 323.2/325.2 [M+H]⁺.

N-(4-(2-chloropyrimidin-4-yl)phenyl)-N-(cyanomethyl)methanesulfonamide(171 mg, 0.53 mmol), 5-amino-2-morpholinobenzoic acid (142 mg, 1.2 eq.)and p-toluene sulfonic acid monohydrate (98 mg, 0.98 eq.) were suspendedin 1,4-dioxane (8 mL) and heated at 100° C. overnight. The reactionmixture was cooled to room temperature, concentrated in vacuo. Theresidue was taken up in ethyl acetate (80 mL) and washed with water (20mL) and brine (20 mL). The organic phase was then dried and concentratedin vacuo. The residue was repeatedly triturated with methanol (5 mL then3 mL) to afford, as a cream solid,5-(4-(4-(N-(cyanomethyl)methylsulfonamido)phenyl)pyrimidin-2-ylamino)-2-morpholinobenzoicacid (101 mg, 37%). ¹H NMR (300 MHz, d₆-DMSO) δ 17.23 (1H, s) CO₂H; 9.99(1H, s); 8.74 (1H, d, J=2.7 Hz); 8.62 (1H, d, J=5.0 Hz); 8.33 (2H, d,J=8.7 Hz); 8.01 (1H, dd, J=2.8, J=8.7 Hz); 7.69 (1H, d, J=9.1 Hz); 7.63(2H, d, J=8.7 Hz); 7.52 (1H, d, J=9.1 Hz); 4.97 (2H, s); 3.81 (4H, m);3.21 (3H, s); 3.06 (4H, m). LC-ESI-MS (method C): rt 5.4 min.; m/z 509.3[M+H]⁺.

5-(4-(4-(N-(Cyanomethyl)methylsulfonamido)phenyl)pyrimidin-2-ylamino)-2-morpholinobenzoicacid (50 mg, 0.098 mmol) andO-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (41 mg, 1.1 eq.) were dissolved in anhydrousN,N-dimethylformamide (4 mL) and sonicated for 5 minutes. Triethylamine(41 mL, 3 eq.) and N,N-dimethylethylenediamine (21 mL, 2 eq.) were addedand the mixture stirred overnight at room temperature. The reactionmixture was then diluted with ethyl acetate (50 mL) and washed withbicarbonate solution (20 mL), water (20 mL) and brine (20 mL). Theorganic phase was dried (sodium sulfate) and concentrated in vacuo toafford, as a yellow solid, Compound 73 (48 mg, 86% yield).

Example 5—Synthesis of Compound 65

To a solution of 5-bromo-2,4-dichloropyrimidine (300 mg, 1.3 mmol) indichloromethane (3 mL) kept at −5° C. was added cold 57% aqueoushydroiodic acid (5 mL). The resulting solution was stirred at −5° C. for2 hours. Solid sodium carbonate was added in small portions until thesolution was pH 7 and the mixture was decolourised by adding 5% aqueoussodium metabisulphite. Water was added until the entire solid dissolvedand the organic phase was separated. The aqueous phase was extractedtwice with dichloromethane then the combined organic layers were driedover anhydrous sodium sulfate, filtered and concentrated to give crude5-bromo-2,4-diiodopyrimidine as a white solid (410 mg). This materialwas used for the next step without further purification. LC-ESI-MS(method B): rt 6.8 min.; m/z 410.9/412.9 [M+H]⁺.

To a mixture of 4-(cyanomethylcarbamoyl)phenylboronic acid (see example3) (185 mg, 0.9 mmol) and 5-bromo-2,4-diiodopyrimidine (410 mg, 1.0mmol) in 1,4-dioxane (10 mL), was added 2M aqueous potassium carbonate(100 μL). The resulting mixture was stirred under nitrogen for 5 minutesthen tetrakis(triphenylphophine)palladium(0) (52 mg, 0.045 mmol) wasadded under a nitrogen atmosphere. The mixture was heated at 80° C.overnight. The cooled reaction mixture was diluted with water andextracted twice with ethyl acetate. The combined organic extracts werewashed with water then brine, dried over anhydrous sodium sulfate,filtered and concentrated to give the crude product as a brown solid.The crude material was purified by flash chromatography, eluting with50% ethyl acetate/petroleum spirit to give4-(5-bromo-2-iodopyrimidin-4-yl)-N-(cyanomethyl)benzamide (200 mg, 35%over 2 steps). LC-ESI-MS (method B): rt 6.2 min.; m/z 443.0/445.0[M+H]⁺.

To a round bottom flask containing4-(5-bromo-2-iodopyrimidin-4-yl)-N-(cyanomethyl)benzamide (45 mg, 0.1mmol) and 4-morpholinoaniline (27 mg, 0.15 mmol) in 1,4-dioxane (3 mL),was added diisopropylamine (26 mg, 0.2 mmol). The flask was equippedwith a reflux condenser and the reaction mixture was heated at refluxovernight. After cooling to room temperature, the reaction mixture wasdiluted with ethyl acetate, washed with water then brine, dried overanhydrous sodium sulfate and concentrated to give the crude product as abrown solid. The crude material was purified by flash chromatography,eluted with 50% ethyl acetate/petroleum spirit then 80% ethylacetate/petroleum spirit to give, as a yellow solid, Compound 65 (12 mg,24%).

Example 6-Salt Formation from Compound 3

Compound 3 (10.0 g) was suspended in methanol (1 L). Concentratedsulfuric acid (10.52 g, 90% w/w) was added dropwise to the stirringsolution. A clear brown solution resulted and a solid lump formed. Thesolution was filtered quickly then allowed to continue stirring for 3 h(a second precipitate appeared within minutes). After this time the paleyellow precipitate was collected by filtration, washed with methanol (10mL) then dried under vacuum overnight to afford4-(4-(4-(4-(cyanomethylcarbamoyl)phenyl)pyrimidin-1-ium-2-ylamino)phenyl)morpholin-4-iumhydrogensulfate, as a pale yellow solid (10.20 g, 69%). m.p. 205° C.Microanalysis: Found C, 45.18; H, 4.36; N, 13.84; S, 10.24.C₂₃H₂₆N₆O₁₀S₂ requires C, 45.24; H, 4.29; N, 13.76; S, 10.50%. ¹H NMR(300 MHz, d₆-DMSO) δ 9.85 (br. s, 1H), 9.34 (t, J=5.4 Hz, 1H), 8.59 (d,J=5.2 Hz, 1H), 8.27 (d, J=8.5 Hz, 2H), 8.03 (d, J=8.5 Hz, 2H), 7.83 (d,J=8.4 Hz, 2H), 7.50 (d, J=5.2 Hz, 1H), 7.34 (br. s, 2H), 4.36 (d, J=5.4Hz, 2H), 3.89 (br. s, 4H), 3.37 (br. s, 4H); ¹³C NMR (75.5 MHz, d₆-DMSO)δ 166.07, 163.36, 159.20, 158.48, 140.19, 139.34, 136.45, 134.89,128.00, 127.22, 121.13, 119.89, 117.59, 109.05, 64.02, 54.04, 27.82.LC-ESI-MS (method D): rt 10.0 min.; m/z 415.1 [M+H]⁺.

Compound 3 (0.25 g) was suspended in methanol (25 ml). Methane sulfonicacid (0.255 g) was added dropwise to the stirring solution and a clearbrown solution resulted. The solution was allowed to stir for 3 h, afterwhich the volume was reduced to 9 ml. The resultant precipitate wascollected and dried under vacuum for 8 h to afford4-(4-(4-(4-(cyanomethylcarbamoyl)phenyl)pyrimidin-1-ium-2-ylamino)phenyl)morpholin-4-iummethanesulfonate as a pale yellow solid (0.22 g). m.p. 208° C. ¹H NMR(300 MHz, d₆-DMSO) δ 9.83 (br. s, 1H), 9.35 (t, J=5.3 Hz, 1H), 8.59 (d,J=5.1 Hz, 1H), 8.28 (d, J=8.5 Hz, 2H), 8.04 (d, J=8.5 Hz, 2H), 7.83 (d,J=9.0 Hz, 2H), 7.50 (d, J=5.5 Hz, 1H), 7.31 (d, J=9.0 Hz, 2H), 4.36 (d,J=5.5 Hz, 2H), 3.88 (m, 4H), 3.35 (br. s, 4H), 2.36 (s, 6H); LC-ESI-MS(method D): rt 10.2 min.; m/z 415.3 [M+H]⁺.

Compound 3 (0.50 g) was suspended in methanol (45 ml). A freshlyprepared solution of hydrochloric acid in methanol (2.6 ml, HCl conc. 40mg/ml) was added dropwise to the stirring solution and a clear brownsolution resulted. The solution was allowed to stir for 2 h, then theresultant precipitate was collected, washed with methanol (5 ml) anddried under vacuum for 8 h to afford4-(4-(4-(4-(cyanomethylcarbamoyl)phenyl)pyrimidin-1-ium-2-ylamino)phenyl)morpholin-4-iumchloride a pale yellow solid (0.30 g). m.p. 210° C. ¹H NMR (300 MHz,d₆-DMSO)¹H NMR (300 MHz, DMSO) δ 9.92 (br. s, 1H), 9.42 (t, J=5.3, 1H),8.62 (d, J=4.8, 1H), 8.29 (d, J=8.1, 2H), 8.06 (d, J=8.1, 2H), 7.89 (d,J=9.0, 2H), 7.53 (br. s, 3H), 4.36 (d, J=5.4, 2H), 3.82 (br. s, 4H),3.43 (br. s, 4H). LC-ESI-MS (method D): rt 10.3 min.; m/z 415.3 [M+H]⁺.

Example 7—Synthesis of Compound 79

A 50 mL two necked round bottom flask was fitted with a magnetic stirrerbar and a dropping funnel. A suspension of NaBH₄ in tetrahydrofuran (100mg, 2.4 mmol/10 mL) was added, followed by5-amino-2-morpholinobenzenecarboxylic acid (222 mg, 1.0 mmol) in oneportion. A reflux condenser was fitted and the reaction mixture wascooled to 0° C. under nitrogen atmosphere. A solution of iodine intetrahydrofuran (250 mg, 1.0 mmol/15 mL) was added dropwise to thereaction mixture. After iodine addition was completed and gas evolutionhad ceased, the reaction mixture was heated at reflux for 6 hours andleft stirring at room temperature overnight. Methanol was added slowlyuntil the mixture became clear. The resulting solution stirred at roomtemperature for 30 minutes then solvent was removed under reducedpressure. The residue was dissolved in 20% KOH (30 mL), stirred for 4hours and extracted with dichloromethane (3×30 mL). The combined organicextracts were washed with brine, dried over anhydrous Na₂SO₄ andconcentrated to give 5-amino-2-morpholinobenzyl alcohol as an off-whitesolid (150 mg, 72% yield). ¹H NMR (300 MHz, CDCl₃) δ 7.05 (d, J=8.7 Hz,1H), 6.59 (dd, J=8.4, 2.7 Hz, 1H), 6.49 (d, J=2.7 Hz, 1H), 5.51 (br s,1H), 4.71 (s, 2H), 3.84 (t, J=5.1 Hz, 4H), 3.60 (br s, 2H), 2.91 (t,J=5.1 Hz, 4H). LC-ESI-MS (method B): rt 2.31 min.; m/z 209.2 [M+H]⁺.

To a suspension of NaH in cold tetrahydrofuran (80 mg/20 mL),5-amino-2-morpholinobenzyl alcohol (400 mg, 2 mmol) was added. Themixture was stirred for 15 minutes then allyl chloride (150 mg, 2 mmol)and tetrabutylammonium iodide (37 mg, 5 mol %) were added. The resultingmixture was stirred at room temperature for 2 hours then at 60° C.overnight. After cooling to room temperature, water was added (200 μL)and the mixture stirred for 10 minutes then diluted with ethyl acetate.The organic phase was washed sequentially with 10% aqueous ammoniumchloride and brine, dried over anhydrous Na₂SO₄, filtered andconcentrated to give a yellow solid. The crude product was purified with50% ethyl acetate in petroleum spirit to obtain3-((allyloxy)methyl)-4-morpholinobenzenamine as a light orange oil (250mg, 50% yield). ¹H NMR (300 MHz, CDCl₃) δ 6.95 (d, J=8.3 Hz, 1H), 6.82(d, J=2.7 Hz, 1H), 6.61 (dd, J=8.2, 2.7 Hz, 1H), 6.10-5.90 (m, 1H),5.34-5.28 (m, 1H), 5.23 (dd, J=10.5, 1.9 Hz, 1H), 4.57 (s, 2H),4.07-4.05 (m, 2H), 3.80 (t, J=4.8 Hz, 4H), 3.55 (br s, 2H), 2.83 (t,J=4.6 Hz, 4H). LC-ESI-MS (method B) rt 5.91 min.; m/z 249.3 [M+H]⁺.

3-((allyloxy)methyl)-4-morpholinobenzenamine was converted to Compound79 by reaction with 4-(2-chloropyrimidin-4-yl)-N-(cyanomethyl)benzamidein the presence of p-toluene sulfonic acid using methods analogous tothose described for the synthesis of Compound 3 and Compound 47.

Compound Analysis

¹H and ¹³C NMR data were acquired on a Brucker AV-300 AVANCE NMRspectrometer.

LC-EI-MS and EI-MS

General Parameters:

LC-EI-MS and EI-MS data were acquired on a Waters 2795 Alliance HPLCcoupled to a Waters 2996 Photodiode Array Detector and Integrity TMDElectron Impact Mass Spectrometer operating under control of WatersMillenium³² software version 4.0 with the settings outlined below.

Mass Spectrometer Parameters:

Helium flow of approximately 0.36 L/min.; acquisition mode set to scan;sampling rate of 1 spectra/sec; source temperature 200° C.; nebulisertemperature 80° C.; expansion region temperature 75° C.; mass range m/z100-550, m/z 100-650 or m/z 100-700 as required.

HPLC Parameters

LC-MS parameters were as described for each of the methods outlinedbelow. EI-MS samples were injected and analysed with no column present,with a solvent flow rate of 0.25 mL/min.

Method A1 (LC-EI-MS)

Solvent Gradient:

% (0.5% aq Time % MilliQ water % ACN formic acid) Curve 0 90 0 10 — 0.590 0 10 6 7.5 0 90 10 6 10.5 0 90 10 6 11.5 90 0 10 6 14.5 90 0 10 6

Flow rate: 0.25 mL/min

Column: one of

-   -   Alltima HP C₁₈ 2.1×150 mm, 5 micron    -   XTerra MS C₁₈, 3.0×100 mm, 3.5 micron    -   XBridge C₁₈, 3.0×100 mm, 3.5 micron

Method A2 (LC-EI-MS)

Solvent Gradient:

Time % MilliQ water % ACN Curve 0 90 10 — 7 0 100 6 9 0 100 6 10 90 10 613 90 10 6

Flow rate: 0.25 mL/min

Column: one of

-   -   Alltima HP C₁₈ 2.1×150 mm, 5 micron    -   XTerra MS C₁₈, 3.0×100 mm, 3.5 micron    -   XBridge C₁₈, 3.0×100 mm, 3.5 micron

LC-ESI-MS

General Parameters:

LC-ESI-MS data was acquired on a Waters 2695Xe HPLC coupled to a Waters2996 Photodiode Array Detector and Waters ZQ Mass Spectrometer operatingunder electrospray ionization conditions with Masslynx software version4.1 with the settings outlined below.

Mass Spectrometer Parameters:

Mass range: m/z 100-650

Scan time: 0.5

Inter scan delay: 0.1

Desolvation gas: 500 L/h N₂

Cone Gas: 100 L/h N₂

Desolvation Temperature: 400° C.

Source Temperature: 120° C.

Cone Voltage: +30 V for ESI positive mode, or

-   -   −45 V for ESI negative mode

HPLC Parameters:

Were one of the following sets of conditions outlined below.

Method B

Solvent Gradient:

Time % MilliQ water % ACN Curve 0 90 10 1 5 0 100 6 6 0 100 6 7 90 10 610 90 10 6

Flow rate: 0.25 mL/min.

Column: XTerra MS C₁₈, 2.1×50 mm, 3.5 micron

Method C

Solvent Gradient:

Time % MilliQ water % ACN % 0.5% formic acid_((aq)) Curve 0 90 0 10 10.5 90 0 10 1 5.5 0 90 10 1 7.5 0 90 10 6 8.5 90 0 10 6 11.5 90 0 10 6

Flow rate: 0.25 mL/min.

Column: XTerra MS C18, 2.1×50 mm, 3.5 micron

Method D

Solvent Gradient:

Time % MilliQ water % ACN Curve 0 90 10 1 10 0 100 6 12 0 100 6 13 90 106 16 90 10 6

Flow rate: 0.25 mL/min.

Column: XTerra MS C18, 3.0×100 mm, 3.5 micron

Example 8—Enzyme Screening

Compound Dilution

For screening purposes, compounds (in 100% DMSO) were warmed at 37° C.for at least 20 minutes before use. A 20 μm stock was initially made inassay buffer, where the final concentration of DMSO was 0.3%. The stockswere then diluted in 384 well Optiplates (Packard) where the finalconcentration of the compound was 5 μM.

JAK Tyrosine Kinase Domain Production

JAK kinase domains were produced using the following procedures:

JAK2

The kinase domain of human JAK2 was amplified from U937 mRNA using thepolymerase chain reaction with the following primers:

SALI-jk2 [SEQ. ID. NO. 7]5′-ACG CGT CGA CGG TGC CTT TGA AGA CCG GGA T-3′ jk2-NOTI[SEQ. ID. NO. 8] 5′-ATA GTT TAG CGG CCG CTC AGA ATG AAG GTC ATT T-3′

The JAK2 PCR products were cloned into the pDest20 destination vector(Gibco). The JAK2 plasmid was then transformed into competent DH10Baccells (Gibco), and the recombinant baculovirus was prepared via Sf9insect cell transfection.

JAK3

The kinase domain of human JAK3 was amplified from U937 mRNA using thepolymerase chain reaction with the following primers:

XHOI-J3 [SEQ. ID. NO. 9] 5′-CCG CTC GAG TAT GCC TGC CAA GAC CCC ACG-3′J3-KPNI [SEQ. ID. NO. 10] 5′-CGG GGT ACC CTA TGA AAA GGA CAG GGA GTG-3′

The JAK3 PCR products were cloned into the pDest20 destinationexpression vector (Gibco). The JAK3 plasmid was then transformed intocompetent DH10Bac cells (Gibco), and the recombinant baculovirus wasprepared via Sf9 insect cell transfection.

Large Scale Production of Kinase Domains

Baculovirus preparations from each of the JAK family members wereinfected into one litre of Sf9 (Spodoptera frugiperda) cells(Invitrogen) grown in SF900II serum free medium (Invitrogen) to a celldensity of approximately 2×10⁶ cells/ml. Cells were infected with virusat a cell culture to virus stock ratio of 20:1. Cells were harvested andlysed 48 hours post infection. The GST-tagged JAK kinase domains werepurified by affinity chromatography on a GSH agarose column(Scientifix).

Assay Protocols

Kinase assays were performed in 384 well Optiplates (Packard) using anAlphascreen Protein Tyrosine KinaseP100 detection kit The compounds werepre-incubated with affinity purified PTK domain in the presence ofphosphotyrosine assay buffer (10 mM HEPES, pH 7.5, 100 mM MgCl₂, 25 mMNaCl, 200 mM sodium vanadate and 0.1% Tween® 20) for 20 minutes. Thecompounds were then incubated with substrate in the presence of either80 or 625 um ATP for 60 or 90 minutes. The substrate used was eithersubstrate-1 with the sequence biotin-EGPWLEEEEEAYGWMDF-NH₂ [SEQ IDNO:13] (final concentration 111 μM) or substrate-2 substrate with thesequence biotin-EQEDEPEGDYFEWLEPE (final concentration 133 μM).Alphascreen phosphotyrosine acceptor beads followed by streptavidindonor beads at a concentration of 1/100 in stop buffer were added toeach well under subdued light and incubated for 2-3 hours. TheAlphascreen plates were read on a Packard Fusion Alpha instrument

The enzyme assay results and structural data for selected compounds isgiven below in Table 2, where +++ is <100 nM, ++ is <500 nM and + is <1μM

Example 9—Cellular Screening

Compound Dilution

For screening purposes, compounds were diluted in 96 well plates at aconcentration of 20 μM. Plates were warmed at 37° C. for 30 minutesbefore the assay was performed.

Establishment of the TEL:JAK2 Cell Line

The coding region encompassing nucleotides 1-487 of TEL was amplified byPCR using the oligonucleotides 5TEL (5′-GGA GGA TCC TGA TCT CTC TCG CTGTGA GAC-3′) [SEQ ID NO 14] and 3TEL (5′-AGGC GTC GAC TTC TTC TTC ATG GTTCTG-3′) [SEQ ID NO 15] and U937 mRNA as a template. A BamHI restrictionsite was incorporated into the 5TEL primer, and a Sal I restriction sitewas incorporated into the 3TEL primer. The regions encompassing thekinase domain of JAK2 (nucleotides 2994-3914; JAK2F 5′-ACGC GTC GAC GGTGCC TTT GAA GAC CGG GAT-3′ [SEQ ID NO 16]; JAK2R 5′-ATA GTT TAG CGG CCGCTC AGA ATG AAG GTC ATT T-3′) [SEQ ID NO 17] and JAK3 (nucleotides2520-3469; JAK3F 5′-GAA GTC GAC TAT GCC TGC CAA GAC CCC ACG ATC TT-3′)[SEQ ID NO 18] were generated by PCR using Taq DNA polymerase(Gibco/BRL) and U937 mRNA as a template. A Sal I restriction site wasincorporated into the forward primer of JAK2 and JAK3, a Not I site wasincorporated into the JAK2 reverse primer and a Xba I site was added tothe reverse primer of JAK3.

A TEL/Jak2 fusion was generated by digestion of the TELPCR product withBamH I/Sal I restriction enzymes, digestion of the JAK2 PCR product withSal I/Not I restriction enzymes, followed by ligation and subcloning ofthe ligation product into the mammalian expression Vector pTRE 2(Clontech), which was prepared by digestion with BamH I-Not Irestriction enzymes, to give the TEL/Jak2 fusion plasmid pTELJAK2.

The TEL/Jak3 fusion was prepared by ligation of the JAK3 Sal I/Not Icleaved kinase domain PCR product with the BamH I/Sal I restrictiondigested TEL product, followed by ligation of the ligation product intothe BamH I/Not I digested pTRE2, to give the TEL/Jak3 fusion plasmidpTELJAK3.

The growth factor dependent myelomonocytic cell line BaF3 bearing thepTET-off plasmid (Clontech) was transfected with either pTELJAK2 orpTELJAK3, and the transfected cells were selected for growth-factorindependent cell growth. The BaF3 wild-type cells were cultured in DMEMcontaining 10% FCS, 10% WEHI 3B conditioned medium. The BaF3 TELJAKcells (BafT_J2 or BafT_J2) were cultured in DMEM 10% Tet-System ApprovedFBS (without WEHI 3B conditioned medium).

Cellular assays were performed as follows:

Cell suspensions were prepared by harvesting cells from culture (thecells used in this test were in late log phase growth with highviability.) Cells were diluted in the appropriate growth medium, asdescribed above, to 1.1× final concentration (from 50,000 cell/mL to200,000 cell/mL, depending on cell line).

Compounds to be tested were added (10 μL, 10× final concentration) to aflat bottomed 96-well plate. The cellular suspension (90 μL per well)was then added, and the plate incubated for 48-72 hr at 37° C., 5% CO₂.Alamar Blue 10 μL per well was added and the plates returned to theincubator for a further 4-6 hours. The plates were then read at 544 nm.

Results

Result are given in table 2 where +++ is <1 μM, ++ is <5 μM and + is <20μM

TABLE 2 Compound JAK2_ JAK3_ BafT_J2_ BAF3wt_ CTLL2_ No. IC50_nM IC50_nMIC50_μM IC50_μM IC50_μM 1 +++ >20 >20 >20 3 +++ ++ +++ ++ ++ 5 ++ >20 NTNT 6 + >20 NT NT 7 >1000 8 ++ 9 ++ ++ ++ ++ ++ 11 +++ ++ ++ ++16 >1000 >1000 >20 >20 >20 17 ++ + ++ + >20 18 ++ + ++ >20 +19 >1000 >1000 >20 20 +++ +++ +++ +++ ++ 21 ++ ++ + + + 22 >1000 ++ >2023 + ++ ++ 25 +++ ++ ++ ++ ++ 31 +++ ++ ++ ++ ++ 34 +++ ++ + ++ ++ 36+++ +++ +++ ++ ++ 39 +++ +++ +++ ++ ++ 42 +++ ++ ++ ++ ++ 44 +++ ++ +++++ ++ 45 +++ +++ +++ +++ +++ 46 +++ +++ +++ ++ +++ 50 +++ + ++ + >20 53+++ ++ +++ ++ ++ 55 +++ +++ +++ ++ ++ 56 +++ +++ +++ ++ ++ 57 +++ + ++++ ++ 58 +++ +++ +++ ++ +++ 59 +++ ++ ++ +++ ++ 65 +++ +++ +++ +++ +++75 +++ + ++ ++ ++ 76 +++ + ++ ++ ++ 79 +++ ++ +++ +++ ++ 80 +++ ++ +++++ ++ 81 +++ >1000 +++ ++ ++ 82 +++ >1000 +++ ++ ++ 85 +++ +++ +++ +++++ 89 +++ ++ ++ ++ ++ 90 +++ ++ ++ +++ + 92 +++ +++ +++ +++ +++ 93 +++++ +++ +++ +++ (NT = Not Tested)

Example 10—Fluorescence Activated Cell Sorter (FACS)

Multiparameter Intracellular Flow Cytometric Analysis of STAT 5Phosphorylation.

The human erythroleukaemic cell line, HEL 92.1.7 (ATCC, TIB-180), wasgrown in RPMI 1640 containing 10% FCS supplemented with 1 mM sodiumpyruvate. For phosphor-STAT 5 determination, HEL cells were grown inRPMI 1640+1% FCS for 18 hours at 37° C. and 2×10⁵ cells per assay pointwere exposed to DMSO/test compounds for 2 hours at 37° C. The cells werecentrifuged at 1300 rpm for 3 minutes and fixed in paraformaldehyde (2%final concentration) for 15 minutes at 37° C. After centrifugation,cells were permeabilized in 90% methanol at 4° C. for 30 minutes.Following three washes in PBS-2% FCS, the staining was performed asfollows using BD PharMingen phycoerythrin-conjugated mouseimmunoglobulin isotype control (Cat. No. 551436 andphycoerythrin-conjugated mouse IgG₁ antibody to STAT 5 (Y694) (Cat. No.612567).

Staining proceeded for 1 hour at room temperature in the dark, followedby 3 washes in PBS-2% FCS. The cells were next resuspended in 800 μLPBS-FCS for FACS analysis. Flow cytometry was performed using a BeckmanCell Lab Quanta SC System with 3 colour and side scatter capabilities.Data analysis was performed with CXP analysis software (version 2.2).The median fluorescence intensity (MFI) was used to determine foldchange upon treatment of cells with specific inhibitor compounds,calculated as the MFI_(stimulated)/MFI_(unstimulated) ratio for thephosphospecific antibody fluorescence channel (FL2).

The results shown in FIG. 2 clearly show a dose-dependent effect onSTAT5 phosphorylation by treatment with compound 3.

Example 11—Western Blots

Experiment 1

Methodology

The murine pro-B cell line BaF3 was routinely maintained in RPMI 1640media containing 10% FCS. On the day of the experiment, cells werewashed twice in PBS, and resuspended in RPMI 1640 media containing 0.1%FCS. After 2 hours of serum deprivation, cells were treated with thedesired concentration of Compound 3, Control Compound, or vehicle alone(DMSO) for a further 2 hours. Mouse IL-3 was then added to cells at afinal concentration of 5 ng/ml for 15 minutes. Cells were then placed onice and washed twice in ice-cold PBS. Washed cell pellets weresnap-frozen in liquid nitrogen and stored at −80° C.

Cell pellets were lysed on ice in RIPA buffer, and lysates clarified bycentrifugation (20,000×g, 4° C., 5 min). The protein concentration oflysates was determined by the Bradford method, and equal amounts ofprotein (60 μg/lane) were separated by SDS-PAGE. Protein was thentransferred to PVDF, and Western blotting performed using an antibodythat specifically recognizes STAT5 phosphorylated at tyrosine 694. Themembrane was then stripped and reprobed with an antibody that recognizestotal STAT5 protein.

The results shown in FIG. 3 clearly show a dose-dependent effect onSTAT5 phosphorylation by treatment with compound 3.

Experiment 2

Methodology

The human erythroleukaemic cell line HEL 92.1.7 was routinely maintainedin RPMI 1640 media containing 10% FCS. The day before the experiment,cells were washed twice in PBS, resuspended in RPMI 1640 mediacontaining 1% FCS, and cultured overnight.

The following day, cells were treated with the desired concentration ofCompound 3, Control Compound, or vehicle alone (DMSO) for 2 hours. Cellswere then placed on ice and washed twice in ice-cold PBS. Washed cellpellets were snap-frozen in liquid nitrogen and stored at −80° C.

Cell pellets were lysed on ice in RIPA buffer, and lysates clarified bycentrifugation (20,000×g, 4° C., 5 min). The protein concentration oflysates was determined by the Bradford method, and equal amounts ofprotein (60 μg/lane) were separated by SDS-PAGE. Protein was thentransferred to PVDF, and Western blotting performed using an antibodythat specifically recognizes STAT5 phosphorylated at tyrosine 694. Themembrane was then stripped and reprobed with an antibody that recognizestotal STAT5 protein.

The results shown in FIG. 4 show a decrease in STAT5 phosphorylationupon treatment with compound 3.

Example 12—Efficacy of Compound 3 on JAK2-Dependent Physiology andTumour Cell Growth

The effect of Compound 3 on growth hormone-stimulated insulin-likegrowth factor-1 concentrations in mouse plasma.

Circulating IGF-1 concentrations (mean±s.e.m.) in female C3/H mice(n=6/group) after administration of compound 3 (50 mg/kg), or vehicleonly (Control, +GH), by oral gavage 8 h and 30 min prior to subcutaneousadministration of growth hormone (+GH, 30 μg/mouse) or saline (Control)at time 0. Blood samples were collected 6 h post −GH administration, andplasma IGF-1 concentrations measured using an ELISA for mouse IGF-1 (R &D Systems). Different superscripts denote significant differences(p<0.05) between groups detected by one-way ANOVA and Bonferroni's testpost-hoc.

The results shown in FIG. 5 show a marked decrease in plasma IGF-1concentration after treatment with compound 3.

Efficacy of orally administered Compound 3 in a subcutaneous tumourmodel of Ba/F3 TelJAK2 cells in nude mice.

Balb/C^(nu/nu) mice were inoculated subcutaneously with mouse Ba/F3TelJAK2 cells (2.5×10⁶/mouse), and dosing b.i.d. by oral gavage withcompound 3 (20 mg/kg, 10 mg/kg, or 5 mg/kg), or vehicle only (5%N-methylpyrrolidone, 0.1 M Captisol®), or Taxol® (5 mg/kg i.v. 3×weekly, n=15 mice/group). Dosing commenced 11 days post-tumour cellinoculation, when tumours were palpable (mean tumour volume of 6 mm³).Tumour dimensions were measured twice weekly. By dosing day 14, the meanpercentage T/C values were 39% for compound 3 at 20 mg/kg b.i.d., 25% at10 mg/kg b.i.d., and 82% at 5 mg/kg/day. Comparison of tumour volumesafter 14 days of dosing by t-test (Mann Whitney Rank Sums Tests) foundsmaller tumour volumes (p<0.05) in groups treated with compound 3 at 20mg/kg b.i.d., and 10 mg/kg b.i.d., and Taxol, compared to the VehicleControl Treated Group and the 5 mg/kg Compound 3 Treated Group, whichwere not different from each other. A more stringent statistical test(Kruskal Wallis One way ANOVA) followed by Dunn's multiple comparisonagainst the Control Group post-hoc identified a significant difference(p<0.05) between the Vehicle Control Treated Group and either theCompound 3 Treated Group (either 10 or 20 mg/kg b.i.d.) or the Taxoltreated Group. The results are shown in FIG. 6.

The results show that compound 3 inhibits the JAK2 enzyme in vitro, aswell as the in vitro growth of Baf3Tel Jak2 cells, which are dependenton constitutively active Jak2 for growth and survival. Baf3Tel JAK2cells growing in vivo as a tumour, are also inhibited by compound 3 in adose-dependent manner. In addition the results demonstrate that compound3 inhibits growth hormone (and therefore JAK2 dependent)-driven IGF-1synthesis and secretion from the mouse liver in vivo.

Example 13—Additional Compound Evaluation

The compounds can also be tested in a murine model ofJAK2^(V617F)-positive myeloproliferative disease (MPD)

Establishment of JAK2^(V617F)-Positive MPD

Bone marrow from male 5-Flurouracil-treated Balb/c mice could beinfected with a JAK2-V617F-GFP retrovirus and retroorbitally injectedinto lethally irradiated female recipients. From day 21 on the micecould be monitored by daily inspection and twice weekly bloodcounts+FACS for GFP-positive cells. It would be expected that a rise inhematocrit could occur around day 28 and a rise of the white blood cellcount around day 40.

Treatment with Compounds

Early intervention group: Treatment would start on day 21 with compoundor carrier given per oral gavage (12 mice in each group). Mice could bemonitored by daily inspection and twice weekly blood counts+FACS forGFP-positive cells. Animals would be sacrificed on day 60 8-12 h afterthe last drug dose. Moribund mice or mice with a white cell count over200,000/nl or weight loss>20% could be sacrificed earlier.

Late intervention group: Groups of 3 mice could be sacrificed on day 29,36, 43, 50 and 57 and bone marrow and spleen could be analyzed forreticulin fibrosis. Treatment could start with compound or carrier givenper oral gavage as soon as fibrosis is documented in 3/3 mice. Micecould be monitored by daily inspection and twice weekly bloodcounts+FACS for GFP-positive cells. Animals could be sacrificed after 30days of therapy 8-12 h after the last drug dose. Moribund mice or micewith a white cell count over 200,000/nl or weight loss>20% could besacrificed earlier. Animals could be subjected to necropsy.

Analysis of Tissues and Survival

Liver and spleen weights could be determined. Tissue sections from bonemarrow, liver and spleen could be analyzed by HE stain. Marrow andspleens could also be silver-stained to assess reticulin fibrosis.Spleen and marrow cells could be analyzed by FACS for GFP, lineagemarkers, JAK2 and STAT5 phosphorylation. Blood could be collected byheart puncture and plasma separated and frozen for drug concentrationmeasurement. Survival between groups could be compared with theKaplan-Meyer method.

Assessment of the Activity of JAK2 Inhibitors in Colony-Forming Assaysof Human Hematopoietic Cells

Peripheral blood mononuclear cells from patients with MPD (predominantlymyelofibrosis) with and without JAK2^(V617F) mutation (N=10 for each)and 5 normal controls (commercial supplier) could be isolated by densitygradient centrifugation (Ficoll). CD34+ cells can be selected usingcommercial kits to enrich for progenitor cells. CD34+ cells can beplated in triplicate in methylcellulose supplemented with fetal bovineserum and cytokines (+/− EPO). After incubation of the plates for 2weeks erythroid and myeloid colony formation could be assessed under aninverted microscope.

Cancer

The effect of the compounds on tumor initiation, progression andmetastasis can be evaluated in relevant in vivo animal efficacy models.Models could be human tumor xenografts models in immuno-deficient mice,from human tumor cell lines or preferably from primary or metastatichuman tumors. Other models might be human tumor xenografts grown inorthotopic sites, models of disseminated disease and transgenic orlabeled tumors models. Models could also include surgical resection ofprimary tumor and evaluation of metastatic disease.

Models could be selected to ensure that the molecular drug targeted isexpressed. Examples of tumors displaying deregulation of the JAK/STATpathway include prostate carcinoma, breast cancer, colon carcinoma,including leukemia, lymphoma, myeloma, ovarian tumors, melanoma, lungcarcinoma, glioma, renal-cell tumors.

Efficacy can be measured in these models by various outcomes dependingon tumor type (solid, leukemia or metastatic) and might include measureof tumor onset, tumor growth rate, tumor burden, tumor growth delay,tumor cell kill, incidence of metastasis, imaging of tumor andinvasiveness/metastasis by various approaches including labeled cells orreagents, survival, angiogenesis, histopathology.

The in vivo animal efficacy models might also be used for determinationof the additivity or synergy of the effect of the compounds incombination with other drugs,

Asthma is restricted to human species, but animal models are often usedto investigate particular aspects of this human disease. Bronchialbiopsies and bronchoalveolar lavage (BAL) fluid recovered from patientswith asthma have been shown to contain an increased number of activatedT cells, B cells, eosinophils and mast cells. Many patients with asthmaare sensitized and have specific immunoglogulin E (IgE) antibodies toone or more inhalant allergens. Atopy is, considered to be a major causeof asthma. In atopic individuals, inhalation of allergens preferentiallyinduces a T-helper 2 cell (Th2) response. In the majority of currentmodels, mice are sensitized by itraperitoneal (ip) injection ofovalbumin (OVA), often together with a Th2 skewed adjuvant, such asalum. In the classical mouse model for asthma, C57/BL6 mice are activelysensitized on day 0 by ip injection of 10 μg of OVA absorbed onto 1 mgof alum. From day 14-21 the mice are exposed daily to aerosolized OVAover a 30 minute period. On day 22, airway inflammation is apparent. BALfluid recovered from these animals demonstrate an increase inperi-bronchiolar space consisting of mixed cellular infiltrates ofmononuclear cells and eosinophils. OVA-specific IgE antibodies can bedemonstrated in the serum of sensitized animals. The mononuclear cellpopulation consists mainly of cells of Th2 phenotype secreting cytokinesIL-4 and IL-5. IL-4 promotes isotype switching of B cells towards IgEsynthesis and IL-5 influences the production, maturation and activationof eosinophils.

PAH

The compounds of formula I can be tested in the dog model of pulmonaryhypertension as described in Gust, R and Schuster, D. P. ExperimentalLung Research, 27:1-12, 2001. They can also be tested in a rabbit modelof monocrotaline induced pulmonary hypertension. The compounds offormula I can also be tested in humans with pulmonary arterialhypertension. The effect of the compounds of formula I can be tested inhumans with pulmonary arterial hypertension by measurement of its acuteeffects on cardiopulmonary hemodynamics. The effect of the compounds onright ventricular pressures, pulmonary artery pressures, pulmonaryvascular resistance, and cardiac output may be determined. The effect ofthe compounds on the six minute walk time, and maximal oxygenconsumption may be determined in humans with PAH. The effect of thecompounds on quality of life (as measured by a questionnaire),hospitalization, and survival may be determined in humans with PAH. Inhumans PAH may be caused by genetic abnormalities (i.e., primary orfamilial PAH) or secondary causes such as scleroderma, uncorrectedcongenital heart disease, mixed collagen vascular disorder, hepatitis C,or other liver disease, HIV infection, or hereditary hemorrhagicteleangiectasia. The effect of the compounds may also be tested on humanendothelial cells, fibroblasts and/or smooth muscle cell lines: forexample, determination of IC50 for STAT3 phosphorylation in humanpulmonary artery smooth muscle cell lines. Cell lines from otherspecies, i.e., the rat may also be examined. The effect of the compoundson precontracted vascular rings from human blood vessels, or bloodvessels from other species, i.e., the rat, may be examined. For example,rat pulmonary artery rings preconstricted with phenylephrine, orendothelin, or serotonin, or vasopressin, angiotensin II, or KCL may bestudied to determine the dose response to the compounds forvasorelaxation. Other vasoconstrictors may be examined.

The effect of the compounds on hypoxia induced pulmonaryvasoconstriction may be examined. A model of hypoxia induced pulmonaryhypertension might include study of rats, such as the Fawn-Hooded ratexposed to low oxygen (i.e., 5 percent oxygen). Another model of hypoxiainduced pulmonary hypertension might include the fetal calf maintainedin a high altitude chamber.

The effect of the compounds may be examined in transgenic models ofpulmonary hypertension: i.e., the BMPR2 knockout mouse treated with IL6,the caveolin1 knock out mouse, or the vasoactive intestinal peptideknockout mouse.

The effect of the compounds on histopathologic changes that occur inboth human and animal models of PAH may be measured. For example, thecompounds may decrease the extent of plexiform lesions in the pulmonaryarterioles of diseased lungs. The plexiform lesion consists ofendothelial cells, smooth muscle cells, and fibroblasts whichproliferate and obstruct to a varying degree, the pulmonary arteriolarlumen.

Example 14—Ex Vivo Analysis of Compound 3 in Cells from JAK2V617FPositive Patients

To assess the activity of small molecule inhibitors of JAK2 an assay hasbeen developed to quantify the activity of the JAK-STAT pathway bymeasuring the phosphorylation status of the downstream protein STAT5.After ligand binding, a haemopoietic cytokine receptor undergoesconformational change activating associated JAK2 protein. Activated JAK2then phosphorylates the intracellular portion of the receptor formingbinding sites for the recruitment of intracellular signaling proteins.STAT5 is one protein that is recruited to the activated cytokinereceptor complex, where it is phosphorylated and then translocates tothe nucleus to regulate the expression of a suite of genes that mediatecellular growth and differentiation.

Intracellular flow cytometry can be used to measure tyrosinephosphorylated STAT5 (pYSTAT5) in specific cell populations by gating onlineage-specific haemopoietic surface markers. This is particularlyimportant for JAK2 V617F positive myeloproliferative disease as theclone containing the mutation only forms a variable fraction of allhaemopoietic cells within the bone marrow. Erythroid cells have beenselected for examination in this study as this lineage is hyperplasticin PV.

Methods

Bone marrow was collected from the ileal crest of patients with JAK2V617F positive myeloproliferative disease. Flow cytometry assays wereperformed on fresh bone marrow samples on the day of the biopsyprocedure. Bone marrow mononuclear cells were collected by densitygradient centrifugation and then 0.75-1.0×10⁶ cells were incubated withcompound 3 at various concentrations for one hour in indicator-free RPMIat 37° C. Cells were maximally stimulated with erythropoietin for 10minutes and then fixed by adding 4% formaldehyde directly into theculture medium. Cells were then permeabilised by cold methanol and thenoptimal concentrations of fluorescent-labeled antibodies added.Erythroid cells were selected for measurement of pYSTAT5 based on cellsurface protein expression (CD45^(lo), CD71^(hi) population).

Results

Compound 3 was tested in the erythroid cell population at varyingconcentrations from 3 μM to 0.0041 μM. The first bone marrow specimenwas examined with a concentration range of inhibitors from 3 μM to 0.037μM. The next two patient specimens were examined with a concentrationrange of between 1 μM and 0.0041 μM.

Unstimulated bone marrow samples with no inhibitor (FIG. 7A—the negativecontrol) showed a variable amount of baseline pYSTAT5 phosphorylationfrom 6 to 32% of the total gated erythroid population. Erythropoietin(EPO) stimulation increased the pYSTAT5 activity in erythroid cells inall specimens examined. This increase in pYSTAT5 with stimulation wasmost apparent in the subset of cells with the highest CD 71 expression(FIG. 7B), consistent with activation of the more immature cells withinthe erythroid population.

All patient samples demonstrated a dose-dependent reduction in STAT5phosphorylation with increasing dose of inhibitor. Results of flowcytometry experiments are presented in two different formats (FIG. 7C).The pYSTAT5 positive population is quantitated as the percentage ofcells in the upper right quadrant of the dot plot graphs (FIGS. 7A andB). The threshold for pYSTAT5 positive events in these graphs is basedon the isotype control antibody staining and was consistent betweenexperiments. Only a subset of the total erythroid population becamepositive with EPO stimulation and this was maximal in the positivecontrol sample. As the dose of inhibitor was increased the number ofpYSTAT5 positive events decreased and this is presented as thepercentage of pYSTAT5 positive events compared to the pYSTAT5 positiveevents in the positive control in the left panel of FIG. 7C. This is arelative measurement within each individual patient specimen.

The second format of presentation is presented in the right panel ofFIG. 7C. This measurement represents the mean fluorescence intensity inthe pYSTAT5 channel and includes both erythroid cells that arestimulated by EPO and those that are not. This is an absolute valuemeasurement of fluorescence and there was variability between thesevalues between the three individuals tested. As the concentration ofinhibitor is decreased the mean fluorescence of the total erythroidpopulation moved towards the value of the positive control.

All publications mentioned in this specification are herein incorporatedby reference. Any discussion of documents, acts, materials, devices,articles or the like which has been included in the presentspecification is solely for the purpose of providing a context for thepresent invention. It is not to be taken as an admission that any or allof these matters form part of the prior art base or were common generalknowledge in the field relevant to the present invention as it existedin Australia or elsewhere before the priority date of each claim of thisapplication.

It will be appreciated by persons skilled in the art that numerousvariations and/or modifications may be made to the invention as shown inthe specific embodiments without departing from the spirit or scope ofthe invention as broadly described. The present embodiments are,therefore, to be considered in all respects as illustrative and notrestrictive.

In the claims which follow and in the preceding description of theinvention, except where the context requires otherwise due to expresslanguage or necessary implication, the word “comprise” or variationssuch as “comprises” or “comprising” is used in an inclusive sense, i.e.,to specify the presence of the stated features but not to preclude thepresence or addition of further features in various embodiments of theinvention.

1-23. (canceled)
 24. A method of treating a hyperproliferative disordercomprising administering to a patient in need thereof a compound of theformula:

or a pharmaceutically acceptable salt thereof.
 25. The method of claim24, wherein the hyperproliferative disorder is cancer.
 26. The method ofclaim 24, wherein the hyperproliferative disorder is acute myelogenousleukemia.
 27. The method of claim 24, wherein the hyperproliferativedisorder is acute lymphocytic leukemia.
 28. The method of claim 24,wherein the hyperproliferative disorder is glioma.